Emerging roles of microRNAs and their implications in uveal melanoma

Abstract

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults with an extremely high mortality rate. Genetic and epigenetic dysregulation contribute to the development of UM. Recent discoveries have revealed dysregulation of the expression levels of microRNAs (miRNAs) as one of the epigenetic mechanisms underlying UM tumorigenesis. Based on their roles, miRNAs are characterized as either oncogenic or tumor suppressive. This review focuses on the roles of miRNAs in UM tumorigenesis, diagnosis, and prognosis, as well as their therapeutic potentials. Particularly, the actions of collective miRNAs are summarized with respect to their involvement in major, aberrant signaling pathways that are implicated in the development and progression of UM. Elucidation of the underlying functional mechanisms and biological aspects of miRNA dysregulation in UM is invaluable in the development of miRNA-based therapeutics, which may be used in combination with conventional treatments to improve therapeutic outcomes. In addition, the expression levels of some miRNAs are correlated with UM initiation and progression and, therefore, may be used as biomarkers for diagnosis and prognosis.

Introduction

Uveal melanoma and differences between uveal and cutaneous melanoma

Uveal melanoma (UM) is the most common adult primary intraocular cancer with an annual incidence of six to seven cases per million [1, 2]. UM and cutaneous melanoma (CM) both derive from melanocytes of the same embryonic origin and cellular function [1]; however, they display remarkable differences in etiology, mutational profile, and clinical progression [2]. Although ultraviolet radiation (UVR) exposure is a proven environmental risk factor for CM, UVR is not involved in UM etiology. Additionally, UM exhibits relatively simple chromosomal alterations, unlike CM, which has very complex cytogenetic alterations. Due to the differences in biological, molecular, and genetic tumor features, the effective clinical therapies for metastatic CM patients are ineffective for metastatic UM patients. The differences between UM and CM have already been comprehensively reviewed by Pandiani et al. [3]. Therefore, the current review will focus on the genetic alterations and abnormal gene expression profiles and signaling pathways in UM. UM mainly originates from melanocytic cells of the choroid (~ 85%), whereas the rest of the cases are found in the iris and the ciliary body [4]. Posterior UM of the ciliary body and choroidal region are more likely to become metastatic due to their location and late detection [5]. Despite good control of the primary UM tumor by surgery or radiation therapy, more than 50% of UM patients will develop metastatic disease within 10–15 years of enucleation [6], and the median survival rate after diagnosis of metastatic UM is 2–9 months [7]. The liver is the most common organ prone to metastasis (89%), followed by the lungs (29%), bones (17%), and skin (12%) [6]. To date, there is not an approved treatment for metastatic UM that can significantly increase the overall survival (OS) rate of patients [1].

Specific gene mutations and dysregulated pathways in UM

Cytogenetic analyses have shown that loss of an entire chromosome 3 homolog (monosomy 3) and an amplification of chromosomal 6p and 8q are the major chromosomal aberrations in UM [8]. UM tumors with monosomy 3 (often associated with amplification of chromosome 8q) are prone to metastasis and have a poor prognosis [9, 10]. Chromosome 3 hosts the tumor suppressor gene BRCA-associated protein 1 (BAP1). The BAP1 gene is located on chromosome 3p21, which encodes an enzyme that is a tumor suppressor protein with deubiquitinase activity and that functions in DNA double-strand break repair [11, 12]. BAP1 mutations occur in approximately 40% of metastatic UM patients [13, 14]. Loss-of-function and/or inactivating mutations of the BAP1 gene are highly associated with metastatic risk [15]. Somatic mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) and guanine nucleotide-binding protein subunit alpha-11 (GNA11) genes are oncogenic drivers in UM. Mutually exclusive mutations in GNAQ or GNA11 are present in over 85% of all examined UM cases [3, 13]. The proteins encoded by the GNAQ/GNA11 genes transduce signals from G protein-coupled receptors (GPCRs) to intracellular pathways [16]. Most of the GNAQ/GNA11 mutations are within the GTPase catalytic domains, resulting in the loss of the GTPase activity. These mutations lead to constitutive activation of downstream signaling pathways, such as PI3K/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and influence melanocyte transformation. The GNAQ/GNA11 mutations activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), indicating that both GNAQ/GNA11 mutants behave as dominant-acting oncogene proteins [14, 17].

Dysregulated pathways downstream of mutant GNAQ/GNA11

RHO/RAC pathway

The members of the RHO family of GTPases are proteins that are involved in a wide variety of cellular functions. Particularly relevant to UM, GNAQ/GNA11 stimulate RAS homolog family member A (RHOA) and RAS-related C3 botulinum toxin substrate 1 (RAC1) and their associated signaling networks through direct activation of triple function domain protein (TRIO) [18–20]. TRIO is a member of the RHO guanine nucleotide exchange factors, and it plays a pivotal role in mediating mitogenic signals in UM. Interestingly, TRIO selectively activates RHOA and RAC1. RAC1 is a small GTP-binding protein that regulates cell proliferation and migration through actin polymerization [21, 22]. The RHO/RAC pathway is activated by oncogenic GNAQ/GNA11 mutations, thus suggesting a new therapeutic target in UM.

MAPK/ERK and PI3K/AKT pathways

Activation of the MAPK/ERK pathway by mutant GNAQ/GNA11 is critical for the development of UM [14]. The incidence of ERK1/2 activation in UM is high and occurs in 86% of primary UM tumors [23]. Mitogen-activated protein kinase (MEK) phosphorylates and activates ERK1/2 and subsequently regulates cellular processes, including proliferation, differentiation, and apoptosis [19, 24]. Another dysregulated pathway in response to GNAQ/GNA11 mutation is the phosphatidylinositol (4,5)-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) pathway. PI3K induces the membrane translocation of AKT to promote cell proliferation and survival [24]. Phosphorylated AKT is observed in approximately 55% of UM cases and is associated with a high risk of metastatic disease [25].

UM and miRNAs

Roles of miRNAs in UM

Recent epigenetic research has elucidated the involvement of microRNAs (miRNAs) in UM [26, 27]. MiRNAs are small (18–22 nucleotides in length), endogenous, noncoding, single-stranded RNAs involved in the regulation of a variety of biological processes, including carcinogenesis. The production of miRNAs involves multiple processing steps that require a large number of molecular events for proper coordination [28]. Gene expression can be altered by miRNAs via binding to the 3′-untranslated region (3′-UTR) of their respective target genes or by destabilizing the messenger RNA (mRNA), the latter of which leads to altered gene expression at the post-transcriptional level [29]. Both oncogenes and tumor suppressor genes are regulated by miRNAs. Thus, a dysregulated miRNA network is a typical feature of many cancers [30]. Emerging evidence has highlighted the implication of miRNAs in UM. Many miRNAs are dysregulated in UM tissues and cell lines, and some of them have been validated as critical regulators in the development of UM [31]. Interestingly, studies have found a link between miRNAs and monosomy 3/BAP1 mutation. Although the major miRNA biogenesis factors are not encoded on chromosome 3, miRNA-processing factors are altered in monosomy 3, regulating the expression levels of certain miRNAs to facilitate metastasis in UM [32]. A miRNA cluster (miR-31-5p, miR-125 family, miR-140-3p, miR-200a-3p, and miR-423-5p) is embedded in the 3′-UTR region of the BAP1 gene. Therefore, BAP1 mutations may contribute to the dysregulated expression of these BAP1-associated miRNAs and their target genes [11]. Elucidation of miRNA target genes, whether oncogenes, tumor suppressor genes, or other cancer-related genes, will explicate how specific miRNAs regulate UM tumorigenesis and provide the basis for novel targeted therapies [33].

UM and OncomiRs

OncomiRs are miRNAs that play crucial roles in the initiation and progression of human cancer. They are generally upregulated in cancers and typically target tumor suppressors to promote tumorigenesis. When oncomiRs are inhibited, tumor cell proliferation, survival, and metastasis can be remarkably reduced. Based on these criteria, several specific oncomiRs have been identified in UM (Table 1), and their target genes and related signaling pathways are summarized in Fig. 1.

Table 1.

Individual upregulated oncomiRs and their target genes in UM

OncomiRs	Expression in UM cell lines and/or UM tumor tissues	Function	Targets	Refs.
miR‑20a	MUM-2B and MUM-2C metastatic UM cells; UM patient tumor tissues	Promotes cell growth, migration, and invasion	N/A	[36]
miR-20a, miR-146a, miR-155, miR-181a, miR-223	MUM-2B and OCM-1 metastatic UM cells	Suppress NK cells, promote metastasis	N/A	[38]
miR-21	OCM-1, M619, and MUM-2B UM cells	Promotes cell proliferation, migration, and invasion	p53	[48]
miR-27a	Genistein-treated human C918 UM cells	Promotes cell growth	ZBTB10	[50]
miR-92a-3p	Decreased expression by histone deacetylase inhibitor MS-275 in OCM-1 UM cells	Decreases susceptibility of UM to TRAIL-mediated apoptosis	MYCBP2	[39]
miR-155	OCM-1A, MUM-2C, C918, and MUM-2B UM cells; UM patient tumor tissues	Promotes cell proliferation and invasion	NDFIP1	[38, 44]
miR-181b	UM cell lines: SP6.5, VUP, OCM-1, 92–1, OCM-1a, and MUM-2b; UM patient tumor tissues	Promotes cell proliferation	CTDSPL	[49]
miR-367	UM cell lines: M17, M23, MUM-2B, and C918; UM patient tumor tissues	Promotes cell proliferation and migration	PTEN	[42]
miR-454	UM cell lines: OCM‐1A, MUM‐2B, MUM‐2C, and C918; UM patient tumor tissues	Promotes cell proliferation and invasion	PTEN	[43]
miR-652	UM cell lines: MUM-2B and MEL270; UM patient tumor tissues	Promotes cell proliferation and migration	HOXA9	[26]

Fig. 1.

Involvement of oncomiRs in UM tumorigenesis via activation of aberrant signaling pathways

The first well-characterized oncomiR is the miR-17–92 polycistronic cluster, which includes miR-17-5p, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a-1 [34, 35]. As a member of the miR-17–92 cluster, miR-20a is significantly upregulated in UM tissues, cell lines, and the plasma of UM patients. Its oncogenic potential has been shown in UM via promotion of cancer cell proliferation and migration [36, 37]. Furthermore, suppression of natural killer (NK) cell activity by miR-20a is implicated in UM metastasis [38]. Another member of the miR-17–92 cluster, miR-92a-3p, is able to reduce the susceptibility of UM cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by directly downregulating MYC-binding protein 2 (MYCBP2) [39].

Aberrant miRNA expression in UM can change the expression of genes in diverse cancer-related signaling pathways. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, functions as downregulator of the PI3K/AKT signaling pathway. PTEN expression is strongly associated with disease-free survival of UM patients [40, 41]. Interestingly, both miR-367 and miR-454 are able to directly downregulate tumor suppressor PTEN expression [42, 43]. In addition, miR-155 interferes with the PTEN and AKT signaling pathways by directly targeting NEDD4-family interacting protein 1 (NDFIP1) [44], which has a key role in the ubiquitination and nuclear translocation of PTEN [45]. Accordingly, high expression and secretion levels of miR-155 are detected in cancer stem cells and circulating CD271⁺ cells that have cancer stem cell features from metastatic UM patients [38], suggesting that the suppression of NK cells by miR-155 may contribute to metastatic progression. Mutation of the tumor suppressor p53 gene is rare in UM, but disruption of the p53 signaling pathway is common [46, 47]. The p53 gene is identified as a direct target of miR-21, which is highly expressed in invasive UM cells. Furthermore, inactivation of p53 and its downstream events by miR-21 lead to more aggressive phenotypes of UM cells [48].

The oncogenic potential of miR-652 has been shown by promotion of hypoxia-inducible factor 1-alpha (HIF-1α) signaling via repression of tumor suppressor homeobox A9 (HOXA9) in UM cells [26]. Overexpression of miR-181b in UM cells promotes cell proliferation through suppression of the target C-terminal domain small phosphatase-like (CTDSPL), which consequently induces the phosphorylation of retinoblastoma (RB, a prototype tumor suppressor) and increases the downstream cell cycle effector transcription factor E2F1 [49]. It is confirmed that miR-27a downregulates the zinc finger and BTB domain-containing 10 (ZBTB10) gene, resulting in inhibition of cell growth [50].

Tumor suppressor miRNAs

Tumor suppressor miRNAs are defined by their properties of downregulating oncogenes [51]. In general, they are often lost or under expressed in cancer cells. GNAQ/GNA11 mutations are the most common somatic mutations occurring in UM, and multiple signaling pathways are induced as a consequence of GNAQ/GNA11 activation [19]. GNAQ/GNA11 and some components of the downstream RHO/RAC, MAPK/ERK, and PI3K/AKT pathways are direct targets of a few tumor suppressor miRNAs. In addition, a number of tumor suppressor miRNAs have been identified by either suppressing cell growth through targeting the genes involved in cell survival signaling pathways or inducing UM cell death through modulating apoptotic signaling (Fig. 2). The expression, function, characterization, and direct gene targets of each individual miRNA are summarized in Table 2.

Fig. 2.

Interference of aberrant UM signaling pathways by tumor suppressor miRNAs

Table 2.

Tumor suppressor miRNAs expression and targets in UM

Tumor suppressor miRNAs	Downregulated in	Functions	Targets	Refs.
let-7b	Radioresistant UM cell lines: OM431 and OCM1	Increases radiosensitivity	cyclin D1	[81]
miR-9	Highly invasive cell lines: MUM-2B and C918	Suppresses cell migration and invasion	NF-κB	[82]
miR-17-3p	UM patient tumor tissues; UM cell lines: OCM-1A, MUM-2C, C918, and MUM-2B	Suppresses tumorigenesis and metastasis	MDM2	[83]
miR-34a	UM cell lines: M17, M21, M23, and SP6.5; UM patient tumor tissues	Inhibits cell proliferation and migration	C-MET	[74]
	UM cell lines: M17, M23, and SP6.5	Inhibits migration and invasion	LGR4	[84]
	UM cell lines: SP6.5 and OM431	Induces apoptosis	TRAIL	[85]
miR-34b/c	UM cell line: SP6.5; UM patient tumor tissues (upregulated in doxorubicin- and epigenetic drug-treated SP6.5 cells)	Inhibits cell proliferation and migration	C-MET	[76]
miR-122	UM cell lines: 92.1, MEL270, OMM2.5, UPMM2, and UPMM3; the TCGA UM dataset, and a cohort of 7 UM patient tumor tissues	Inhibits cell proliferation and migration	C-MET, ADAM10	[77]
miR-124a	UM cell lines: M17, M21, M23, SP6.5; UM patient tumor tissues	Inhibits cell proliferation and invasion	CDK4, CDK6, cyclin D2, EZH2	[86]
miR-137	UM cell lines: OMM1.3, Mel202, 92.1, OMM1, OCM1, and OCM3	Inhibits cell proliferation	p160SRC3	[87]
	UM cell lines: SP6.5 and OM431	Induces apoptosis and	TRAIL	[85]
	UM cell lines: M17, M23, and SP6.5	Inhibits cell proliferation	MITF, CDK6	[88]
miR-142-3p	UM cell lines: M17, SP6.5; UM patient tumor tissues	Suppresses cell proliferation and migration	CDC25C, TGFβR1, GNAQ, WASL, RAC1	[52]
miR-144	UM cell lines: MUM-2B, C918, MUM-2C, and OCM-1A; UM patient tumor tissues	Inhibits cell proliferation and invasion	C-MET	[75]
	UM cell lines: 92.1, MEL270, OMM2.5, UPMM2, and UPMM3; The TCGA UM dataset and a cohort of 7 UM patient tumor tissues	Inhibits cell proliferation and migration	C-MET, ADAM10	[77]
miR-145	UM cell lines: MUM-2B and OCM-1; UM patient tumor tissues	Inhibits cell growth and promotes cell apoptosis	IRS-1	[65]
	UM patient tumor tissues (both high- and low-invasive UM)	Inhibits proliferation and invasion	NPR1	[60]
miR-182	UM cell lines: M23 and SP6.5; UM patient tumor tissues; (upregulated by p53 activation)	Decreases cell growth, migration, and invasion	MITF, BCL2, cyclin D2	[80]
	UM cell lines: SP6.5 and OM431	Induce apoptosis	TRAIL	[85]
miRNA-205	UM patient tumor tissues (both high- and low-invasive UM)	Inhibits proliferation and invasion	NPR1	[60]
miR-216a-5p	UM cell line: MUM-2B; UM patient tumor tissues	Inhibits cell growth through the Warburg effect	HK2	[69]
miR-224-5p	UM cell line: OCM-1A; UM patient tumor tissues	Inhibits proliferation, migration, and invasion,	PIK3R3/AKT3	[62]
	UM cell lines: OCM-1A, MUM-2C, C918, and MUM-2B; UM patient tumor tissues	Suppresses cell proliferation and migration	FTH1P3, RAC1, Fizzled 5	[58]
miR-15a, miR-185, miR-211	Cell line: OCM-1; UM patient tumor tissues	Inhibit cell proliferation	IL-10Rα	[67]

RHO/RAC pathway-targeting miRNAs

Peng et al. showed that miR-142-3p regulates GNAQ/GNA11 and the downstream RHO/RAC signaling pathway as well as components of the MAPK/ERK and PI3K/AKT pathways. GNAQ, RAC1, transforming growth factor beta receptor 1 (TGFβR1), cell division cycle 25C (CDC25C), or Wiskott–Aldrich syndrome protein (WASL) are target genes of miR-142-3p [52, 53]. TGFβR1 functions as a transducer and acts as an oncogene in UM. CDC25C is a phosphatase that triggers entry into mitosis by dephosphorylating cyclin B-CDK1 [54–56]. WASL holds key roles in the regulation of endocytosis, actin polymerization, and cell junction formation [57]. Based on their functions, downregulation of the expression of these five genes accounts for decreased cell proliferation and migration [52]. RAC1 is also a direct target of miR-224-5p. Additionally, miR-224-5p targets frizzled-5, a receptor protein in the WNT/β-catenin pathway, which is activated in many cancers and is believed to contribute to tumor recurrence [58, 59]. A recent study suggested that miR-145 and miR-205 function as onco-suppressors in UM by directly targeting neuropilin 1 (NRP1) to reduce the expression of human cell division control protein 42 (CDC42), a small GTPase of the RHO family [60]. The homologous RHO GTPases, RAC, and CDC42, play key roles in regulating the occurrence and development of tumors by controlling diverse cellular functions [61]. NRP1 is a transmembrane coreceptor for both vascular endothelial growth factor (VEGF) and semaphorin family members. It also interacts with various membrane receptors, such as C-MET, integrins, and transforming growth factor receptors to promote angiogenesis, tumor growth, invasion, and metastasis.

PI3K/AKT and MAPK/ERK pathway-targeting miRNAs

The PI3K/AKT signaling pathway is activated in a setting of GNAQ/GNA11 mutation and is involved in promotion of cell survival and cell migration in UM [24]. Tumor suppressor miR-224-5p directly targets PI3K/AKT pathway-related proteins, such as phosphatidylinositol 3-kinase regulatory subunit gamma (PIK3R3) and AKT3 (protein kinase B gamma), to exert its inhibitory effects on UM cell growth and invasion [62]. Insulin receptor substrate-1 (IRS-1) is an oncogene that is implicated in the upregulation of the PI3K/AKT pathway [63, 64]. Cell proliferation is inhibited and UM cell death is promoted by miR-145 through targeting IRS-1 [65]. IL-10, an immunoregulatory cytokine, can induce phosphorylation of AKT to active PI3K/AKT signaling [66]. The expression of IL-10 receptor (IL-10Rα) in UM is downregulated by miR-15a, miR-185, and miR-211, individually and in combination [67]. Single or combined ectopic expression of these three miRNAs leads to a significant reduction in UM cell proliferation [67]. The expression of hexokinase-2 (HK2) is induced by activated PI3K/AKT signaling [68], and it is the primary rate-determining enzyme involved in aerobic glycolysis, which preferentially occurs in cancer cells and is referred to as the Warburg effect. UM growth is reduced by miR-216a-5p through suppression of the Warburg effect by directly targeting HK2 [69].

The C-MET tyrosine-kinase receptor is implicated in the upstream pathway of PI3K/AKT [70, 71]. In UM, C-MET is overexpressed in over 60% of tumors, and it is associated with tumor aggression and metastasis [72, 73]. To date, several miRNAs have been identified that regulate C-MET expression, such as miR-34, miR-122, miR-144, and miR-182 [74–77]. The members of the miR-34 family are miR-34a, miR-34b, and miR-34c. Although three of them are encoded by two different transcriptional units, their sequences are very similar [78]. They act as tumor suppressors by abrogating several signaling pathways involved in cell proliferation and migration [74, 76]. They downregulate C-MET expression, which in turn interferes with the downstream AKT signaling pathway in an hepatocyte growth factor (HGF)-dependent manner [70]. The expression of C-MET can be directly and indirectly downregulated in UM by miR-122 and miR-144 [75, 77] because they both directly target disintegrin metalloproteinase 10 (ADAM10), a zinc‐dependent transmembrane protein with pro-invasive roles in UM cells. ADAM1 is associated with UM progression by cleaving c-Met into soluble c-Met [79]. Nevertheless, miR-182 interferes the C-MET signaling pathway by targeting melanogenesis associated transcription factor (MITF) [80].

Microphthalmia-associated transcription factor (MITF) encodes a basic helix-loop-helix leucine zipper transcriptional factor and is an oncogene that is associated with the AKT and ERK1/2 pathways in UM. C-MET is a direct transcriptional target of MITF [89], and the latter is targeted by miR-182 in UM cells. Furthermore, UM cell growth is suppressed by miR-182 via the binding of MITF, cyclin D2, and B-cell lymphoma 2 (BCL2) [80]. BCL-2 is a mitochondrial protein that promotes cellular survival and inhibits the activity of pro-apoptotic proteins [80]. Additionally, miR-182 participates in the tumor suppression network of p53 in UM because miR-182 expression depends on p53 activation [80]. Similar to the function of miR-182, miR-137 suppresses the expression of MITF and cell cycle-related genes. The expression of miR-137 is epigenetically downregulated during UM tumorigenesis [85, 88]. Functional analysis has indicated that miR-137 significantly increases G1 cell cycle arrest of UM cells by suppressing the expression of MITF, cyclin-dependent kinases CDK2 and CDK6, and three p160 steroid receptor coactivators (SRCs) [87]. Of special note, SRCs are transcriptional coactivators and “master regulators” of genes involved in tumorigenesis.

NF-κB signaling pathway interference by miRNAs

The aggression of UM is believed to be associated with the nuclear factor kappa B (NF-κB) pathway. NF-κB1 is known to play key roles in cell proliferation, tumoral angiogenesis, and metastasis. Liu et al. reported that miR-9 downregulates the expression of NF-κB1 and its downstream targets, such as matrix metalloproteinases-2 and -9 (MMP-2, -9) and VEGFA [82]. MMP-2- and MMP-9-mediated extracellular matrix degradation is essential for cellular invasion and cancer cell metastasis [90]. MMP-2 is a downstream effector for leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). LGR4 is involved in tumor metastasis, and miR-34a targets LGR4 to regulate the migration and invasion of UM cells [84, 91].

P53 and other cell cycle-related pathway-targeting mRNAs

The p53 pathway is central among the molecular mechanisms that influence cell cycle arrest and apoptosis [92]. Nonetheless, the retinoblastoma tumor suppressor protein (pRB) is a key player in cell cycle progression, and one of its major functions is to prevent excessive cell growth. However, the RB pathway is frequently dysregulated in many cancers [93]. The canonical RB pathway consists of RB1, cyclin D1, cyclin-dependent kinase 4/6 (CDK4/6), p16, and the E2F family. The principal function of CDK4/6 is to phosphorylate pRB and consequently control G1/S progression. Cancer cells often restrain the inhibitory function of pRB on cell cycle progression via CDK4/6 [94]. It has been reported that miR-17-3p increases the transcriptional activity of p53 by downregulating the expression of oncoprotein murine double minute clone 2 (MDM2) [83], which mediates the proteasomal degradation of p53 through its E3 ligase activity [95]. UM cell proliferation is reduced by miR-34a and miR-34b/c through indirect modulation of the cell cycle proteins RB, CDC2, E2F3, and CDK4/6 [74, 76]. It has been shown that miR-124a suppresses the functions of CDK4/6, cyclin D2, and EZH2, and it also exhibits strong inhibitory effects on UM cell migration and invasion as well as tumor growth in vivo [86].The expression of miR-124a is epigenetically silenced in UM development, and its expression can be restored by treatment with a DNA-hypomethylating agent or histone deacetylase inhibitor [86]. Let-7b is a well-known tumor suppressor miRNA and is downregulated in radioresistant UM cells [96]. Direct targeting of cyclin D1 leads to increased radiosensitivity of UM cells by let-7b [81].

In summary, some miRNAs can influence UM tumorigenesis and metastasis and are potential targets for the development of novel therapeutic strategies. A specific miRNA may have diverse roles in different subtypes of UM because a single miRNA can target multiple genes to interact with several signaling pathways. Therefore, interpretation of the effects of specific miRNAs in UM should be relevant to the biological backgrounds.

Biomarker miRNAs for diagnosis and prognosis of UM

Efforts to understand the molecular biology of UM have revealed some miRNAs that correlate with patient prognoses [97]. In UM, miRNAs can be detected and quantified in tumor tissues [frozen and formalin-fixed, paraffin-embedded tissues (FFPE)] and blood. Particular miRNA signatures appear different from healthy cells [5, 98]. The data summarized here may help guide UM biomarker development to identify metastatic UM (Table 3).

Table 3.

Summary of miRNAs involved in human UM detection

Samples from UM patients	Techniques for miRNA detection	Overexpressed underexpressed in primary UM	Overexpressed underexpressed in metastasis/high metastatic risk UM	Refs.
Tumor tissues	miRNA microarray chips; qPCR	miR-17, miR-20a, miR-21, miR-34a, miR-106a, miR-145, miR-204		[99]
Tumor tissues	miRNA sequencing		miR-16-5p, miR-17-5p, miR-21-5p miR-99a-5p, miR-99a-3p, miR-101-3p, miR-132-5p, miR-151a-3p, miR-181a-2-3p, miR-181b-5p, miR-378d, miR-1537-3p, let-7c-5p	[102]
Tumor tissues	Agilent miRNA microarray		let-7b, miR-143, miR-193b, miR-199a, miR-199a*, miR-652	[105]
FFPE tumor tissues	miRNA Agilent arrays- hybridization	miR-1, miR-10a, miR-18a, miR-19b-1*, miR-26a-2, miR-34c-5p, miR-129*, miR-133a, miR-154, miR-181a*, miR-218, miR-369-3p, miR-377, miR-376c, miR-493*, miR-495, miR-586, let-7e	miR-33a, miR-99a*, miR-135b, miR-196a*, miR-325, miR-497*, miR-512-5p, miR-549, miR-556-5p miR-585, miR-640, miR-885-5p	[100]
FFPE tumor tissues	miRNA Agilent microarray	miR-134, miR-143, miR146b, miR-199a, miR-214	miR-134, miR-149*	[104]
FFPE tumor tissues	Tissue microarrays (TMA); qPCR		miR-592, miR-346, miR-1247; miR-506, miR-513c	[106]
TCGA UM database	large-scale genome sequencing		miR-195, miR-224, miR-365a, miR-365b, miR-452, miR-513c, miR-873, miR-4709, miR-7702	[108]
TCGA UM database	Illumina Hiseq		miR‑592, miR‑199a‑5p; miR-211-5p, miR-514a-3p, miR-508-3p, miR-509–3-5p, miR-513a-5p, miR-513c-5p	[107]
Serum	MELmiR-17 panel qPCR	miR-16, miR-145, miR-146a, miR-204, miR-211, miR-363-3p	miR-211	[5]
Plasma, tumor tissues	Illumina microRNA profiling BeadChip, quantitative nuclease protection assay, qPCR		miR-199-5p, miR-223, miR-92b	[32]
Serum	qPCR	miR-20a, 125b, 146a, 155 and 223, miR-181a	miR-20a, 125b, 146a, 155 and 223, miR-181a	[37]
Vitreous humor, serum, vitreal and serum exosomes	TaqMan low density array	Vitreous humor and vitreal exosomes: miR-21, miR-34a, miR-146a, serum and serum exosomes: miR-146a		[110]
Serum, FFPE tumor samples	TaqMan low density array, TaqMan miRNA assay	Serum: miR-146a, miR-523; miR-19a, miR-30d, miR-127, miR-451, miR-518f, miR-1274B FFPE tumor samples: miR-146a		[111]

Detection of miRNAs in UM tissues and cells

Comparison of the miRNA-expression profiles of UM and normal uveal tissues revealed that miR-17, miR-20a, miR-21, miR-34a, and miR-106a are upregulated, whereas miR-145 and miR-204 are downregulated in UM. Thus, it is suggested that differentially expressed miRNAs may help in UM diagnosis [99]. Radhakrishnan et al. performed miRNA-expression profiling on FFPE sections of UM tumors and found that 18 miRNAs are differentially expressed in non-metastatic UM, whereas 12 miRNAs are differentially expressed in metastatic UM [100]. Londin et al. also reported that miRNA isoforms are associated with UM metastasis and patient survival [101]. After analyzing the expression of miRNAs in 26 human UM samples, Smit et al. identified 13 differentially expressed miRNAs and three known oncomiRs (miR-17-5p, miR-21-5p, and miR-151a-3p) that are overexpressed in high-risk patients (with an average disease-free survival rate of 28 months, a BAP1 mutation, and BAP1-negative immunoreactivity). This study suggests that some miRNAs play key roles in the development of UM metastasis [102]. Thus, further validation of these findings is warranted to explore the potential role of miRNAs as biomarkers in UM tumor progression and metastasis In UM, alterations on chromosome 3 (a predictor of metastasis) have been linked to metastatic death [103], and links between miRNAs and clinicopathological features in UM with monosomy 3 or disomy 3 have been studied by several research groups. Using univariate and multivariate analyses of miRNA expression in FFPE UM samples [104], Venkatesan et al. discovered that five miRNAs (miR-134, miR-143, miR-146b, miR-199a, and miR-214) have different expression patterns in monosomy 3/disomy 3 UM tumors in a South Asian Indian cohort. The expression of miR-134 and miR-149-3p are strongly correlated with liver metastasis [104]. Worley et al. screened differentially expressed miRNAs using the Agilent miRNA microarray platform to predict metastatic risk and revealed that expression of a group of miRNAs (let-7b, miR-143, miR-193b, miR-199a, miR-199a-3p, and miR-652) is upregulated in metastatic UM tumors and is associated with chromosome 3 status. Two miRNAs (let-7b and miR-199a) are validated as the most significant discriminators for metastatic UM [105]. Wróblewska et al. reported significant upregulation of miR-346, miR-592, and miR-1247, but downregulation of miR-506 and miR-513c, in tumors of metastatic UM patients. Moreover, miR-592 expression is correlated with monosomy 3 tumors [106]. Overall, these independent studies strongly suggest that the expression pattern of miRNAs can be used to stratify UM and to predict metastasis and overall survival of UM patients.

In recent years, different computational approaches have been used to analyze the miRNA-expression data in the TCGA (The Cancer Genome Atlas) UM database to identify miRNAs as prognostic biomarkers. Using this approach, Falzone et al. found that five miRNAs of the miR‑506‑514 cluster (including miR-508-3p, miR-509-3-5p, miR-513c-5p, miR-513a-5p, and miR-514a-3p) are downregulated, whereas miR‑592 and miR‑199a‑5p are upregulated in UM patients with high grade or high risk of metastatic disease. The downregulation of miR-211-5p plus the five miRNAs of the miR‑506‑514 cluster are significantly associated with a negative prognosis in UM patients [107]. Nonetheless, Xin et al. analyzed the miRNA-expression profiles of 80 UM patients from the TCGA database and identified a signature of nine miRNAs (miR-195, miR-224, miR-365a, miR-365b, miR-452, miR-513c, miR-873, miR-4709, miR-7702) for the prognosis of UM. This set of miRNAs can be used to distinguish high-risk UM patients with significantly shorter overall survival rates from those in the low-risk group [108]. Taken together, these preliminary findings need to be further validated by in vitro and translational approaches to develop specific miRNAs as diagnostic/prognostic biomarkers for the management of UM.

Contrary to the aforementioned findings, Larsen et al. performed multiple analyses to determine the association of miRNA expression and chromosomal changes with metastasis and patient survival in UM. They analyzed the miRNA expression of 13 metastatic UM patients compared with that of 13 age‐ and gender‐matched non-metastatic UM control patients, but they did not find miRNAs related to metastasis or overall survival. The prognostic value of miRNA expression has not been confirmed in this study [109].

Circulating biomarker miRNA

Circulating miRNAs are determined to be eminently specific and sensitive for identifying UM malignancy. They are expected to become a new type of blood biomarkers for the diagnosis and prognosis of UM [5, 33, 98, 110, 111]. A group of six miRNAs (miR-16, miR-145, miR-146a, miR-204, miR-211, and miR-363-3p) are differentially expressed in the sera of UM patients and patients with benign melanocytic lesions of the posterior uvea (known as choroidal nevi). Notably, miR-211 is differently expressed in both metastatic and localized UM [5]. Achberger et al. observed high levels of miR-20a, miR-125b, miR-146a, miR-155, miR-181a, and miR-223 in the sera of UM patients. When metastasis occurs in these patients, levels of these miRNAs increase, but miR-181a levels decrease [37]. Russo et al. analyzed the serum levels of miRNAs and found eight miRNAs that are differentially expressed in UM patients compared to normal controls; miR-146a and miR-523 are upregulated, whereas miR-19a, miR-30d, miR-127, miR-451, miR-518f, and miR-1274b) are downregulated. Importantly, the level of miR-146a is increased in both serum and FFPE UM samples [111]. Ragusa et al. also reported the upregulation of miR-146a in the sera, serum exosomes, and FFPE samples of UM patients [110]. Remarkably, miR-146a is upregulated in the four aforementioned investigations, which strongly suggests that miR-146a is a potential circulating marker of UM. Triozzi et al. investigated the plasma levels of miRNAs in UM patients with either tumor monosomy 3 or disomy 3 compared with normal controls. They noticed that miR-92b, miR-199a-5p, and miR-223 are specifically overexpressed in patients with monosomy 3 [32].

Extracellular vesicles and miRNAs

Extracellular vesicles (EVs) include exosomes (size ranges from 30 to 100 nm), microparticles (size ranges from 100 to 1000 nm), and other cell-derived membranous structures. EVs contain various proteins, lipids, metabolites, and nucleic acids, and they transfer bioactive molecules from their parental cells to recipient cells to facilitate intercellular communication. It has been shown that EVs transfer miRNAs into target organs or cells and play a key role in tumorigenesis, metastasis, and therapeutic responses [33]. EVs are also present in many human bodily fluids and have been tested as markers for the diagnosis and prognosis of different diseases [112, 113]. Eldh et al. showed that patients with liver metastases have significantly more exosomes in their peripheral blood compared with healthy controls, and the exosomes from liver perfusates contain different miRNA clusters compared with the exosomes released from other types of cancer cells [114]. Ragusa et al. analyzed the miRNA profiles of exosomes from vitreous humor (VH) and serum exosomes of UM patients. They found high levels of miR-21, miR-34a, and miR-146a in the exosomes from VH and high levels of miR-146a in the serum exosomes, which suggests that these miRNAs may be released by the eyes affected by UM and could be considered as potential circulating biomarkers of UM [110].

Among these studies, only a few miRNAs (e.g., miR-146, miR-199a) are common, and some miRNAs, such as miR-181a, exhibit discordant expression patterns. Possible reasons for these discrepancies are discussed in a very recent review article [33]. Bande Rodriguez et al. suggest the following possible causes: UM sample quality, classification and inclusion criteria, clinical treatments, sample processing, tumor heterogeneity, different racial groups, miRNA quantification methods, and other patient clinical features. All these differences should be considered during interpretation of the data. Although using blood miRNA biomarkers is a less invasive diagnostic method, varied results have been generated from different studies. In the future, unification of the criteria for inclusion and exclusion of specimen collection and standardization of analysis methods is needed to produce more reliable data.

Therapeutic use of miRNAs for UM treatment

Current therapeutic approaches of chemotherapies or targeted therapies yield very low response rates for metastatic UM [115]. For example, selumetinib selectively targets the MAPK pathway, and AZD 8055 interferes with the mammalian target of rapamycin (mTOR) signaling pathway in UM cells; however, they both target these same signaling pathways in healthy cells as well. Emerging studies suggest a key role of a number of miRNAs in UM progression and that some specific miRNAs are involved in chemotherapeutic resistance [116]. Despite having potential to be developed as therapeutic tools, some disadvantages of miRNAs reduce their therapeutic activity. For example, miRNAs have very low stability because they can be degraded by enzymes circulating in the blood [117]. They also have a low endocytosis rate due to their negative charge [118]. Finally, they non-selectively target both healthy and cancer cells [117]. To overcome these limitations, nanoparticle delivery systems have been developed. Nanocarriers include liposomes, viral vectors, polymeric or peptide nanoparticles, lipid nanoparticles, etc. [119–124]. Rois et al. successfully used gold nanoparticles (AuNPs) as carriers to improve the stability and internalization of four miRNAs and chemotherapeutic SN38. Furthermore, the four miRNAs (miR-34a, miR-137 miR-144, and miR-182) were chosen based on their downregulation in UM, and their combined application exhibited a synergistic effect [125]. Moreover, SN38 (7-ethyl-10-hidroxycamptothecin) is a topoisomerase I inhibitor with poor aqueous solubility [126], and the combination of these four miRNAs increases UM cell sensitivity to chemotherapeutic SN38 [116]. Remarkably, these studies demonstrate that the conjugated effect of the four miRNAs and SN38 with AuNP delivery overcomes the innate constraints of these individual molecules and proves to be a highly effective therapeutic approach against UM [116].

Perspectives

Future studies are expected to explicate the involvement of miRNAs in UM tumorigenesis and resistance to chemotherapy and radiation. Proper interpretation of the effects of specific miRNAs in UM progression requires consideration of the appropriate biological backgrounds and further verification with larger sample sizes. The use of well-developed nanotechnology drug-delivery systems may overcome the limitations of therapeutic miRNAs (miRNA mimics and inhibitors of miRNA) and chemotherapy. Combination of conventional therapies with miRNA-based strategies may allow more effective targeting of UM cancer cells and minimize the risk of patient relapse. Besides their therapeutic potential, miRNAs can be developed as biomarkers for diagnosis and prognosis because unique miRNA profiles are associated with specific tissues and cancer stages of UM.

Funding Statement

This work was supported by the Canadian Institutes of Health Research under an operating grant [362383].