The role of microRNAs in bladder cancer.

Bladder cancer (BC) is the fifth most common cancer worldwide and is associated with significant morbidity and mortality. The prognosis of muscle invasive BC is poor, and recurrence is common after radical surgery or chemotherapy. Therefore, new diagnostic methods and treatment modalities are critical. MicroRNAs (miRNAs), a class of small noncoding RNAs, regulate the expression of protein-coding genes by repressing translation or cleaving RNA transcripts in a sequence-specific manner. miRNAs have important roles in the regulation of genes involved in cancer development, progression, and metastasis. The availability of genomewide miRNA expression profiles by deep sequencing technology has facilitated rapid and precise identification of aberrant miRNA expression in BC. Indeed, several miRNAs that are either upregulated or downregulated have been shown to have associations with significant cancer pathways. Furthermore, many miRNAs, including those that can be detected in urine and blood, have been studied as potential noninvasive tumor markers for diagnostic and prognostic purposes. Here, we searched PubMed for publications describing the role of miRNAs in BC by using the keywords "bladder cancer" and "microRNA" on March 1, 2016. We found 374 papers and selected articles written in English in which the level of scientific detail and reporting were sufficient and in which novel findings were demonstrated. In this review, we summarize these studies from the point of view of miRNA-related molecular networks (specific miRNAs and their targets) and miRNAs as tumor markers in BC. We also discuss future directions of miRNA studies in the context of therapeutic modalities.

INTRODUCTION

Bladder cancer (BC) is one of the leading causes of cancer-related death worldwide, with an estimated 429,800 new cases and 165,100 deaths globally in 2012 [1]. BCs can be categorized into 2 groups: nonmuscle invasive BC (NMIBC) and muscle invasive BC (MIBC). Approximately 70% to 80% of patients are diagnosed with NMIBC, and some of these patients have a high risk of recurrence and a variable risk of progression despite administration of local therapies [2]. The remaining 25% of patients with MIBC are managed by radical surgery or radiotherapy, but often still have poor outcomes, despite administration of these systemic therapies [2]. For patients with advanced BC or failure of first-line chemotherapy, no clearly defined second-line treatments have been shown to prolong overall survival [2]. Therefore, elucidation of novel molecular mechanisms of BC progression and identification of novel tumor markers that can predict recurrence or survival are urgently needed.

Despite its relatively low sensitivity (30%–40%), urinary cytology is a reliable marker for BC diagnosis because of its high specificity (90%–95%). However, because of the low sensitivity of urinary cytology, patients are still forced to undergo a painful cystoscopic procedure to confirm the initial diagnosis and to check for recurrence during follow-up examinations after undergoing transurethral resection of bladder tumors. Several new urine-based tests for BC, such as bladder tumor antigen, nuclear matrix protein 22 (NMP22, Sysmex Corp., Kobe, Japan), and FISH (UroVysion, Abbott Molecular Inc, Des Plaines, IL, USA), have been approved for clinical use. However, these new urinary markers have not been widely applied because they have relatively low specificities (60%–80%) compared with urinary cytology, despite their higher sensitivities (50%–70%) [3]. Thus, specificity may come at the cost of sensitivity, and conventional urinary cytology is a good example of this compromise. Accordingly, new effective diagnostic markers are urgently needed for patients with BC.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules (19–22 bases in length) that negatively regulate the expression of protein-coding genes in a sequence-specific manner [4]. The nature of miRNAs is unique in that one miRNA can regulate multiple protein-coding RNAs. Bioinformatics predictions have indicated that miRNAs regulate 30% to 60% of the protein-coding genes in the human genome [56]. Growing evidence has demonstrated that aberrantly expressed miRNAs can act as oncogenes or tumor suppressors in various types of malignancies [7]. These miRNAs can disrupt tightly controlled RNA networks in cancer cells [8]. Since 2006, we have rigorously investigated the molecular networks of miRNAs and their target genes in clinical BC. Identification of aberrantly expressed miRNAs in BC could provide important clues for the investigation of novel molecular mechanisms of initiation, progression, and metastasis in BC. In this review, we discuss the role of miRNAs in BC on the basis of up-to-date results from our laboratory and other research groups.

CURRENT CONSENSUS OF EPIGENETIC GENE REGULATION BY NONCODING RNA

After the completion of the Human Genome Project in 2003, about 20,000 to 25,000 genes were identified, and the sequences of 3 billion base pairs of human DNA were determined [9]. Surprisingly, only 2% of the human genome encodes functional proteins; 50% to 75% of the human genome is transcribed, and 98% of the transcripts are not translated into protein. Fig. 1 shows the current consensus of epigenetic regulation by noncoding RNAs transcribed from approximately 70% of the genomic region that used to be considered as "junk DNA" [10]. These noncoding RNAs can be roughly classified into two groups according to size. The first group includes short RNAs of less than 200 nucleotides (nt) in length; miRNAs, which are small RNA molecules of around 18 to 22 nt in length, fit into this group, as do other classes of small RNAs, such as piwi-interacting RNAs (around 23–30 nt) [11]. The other group includes long noncoding RNAs (lncRNAs) of around 200 nt or more. lncRNAs have been implicated in a range of developmental processes and diseases and can inhibit or activate gene expression through a range of diverse mechanisms. In contrast to miRNAs, which have been extensively studied and have well-understood functions in gene regulation, lncRNAs are not well studied or characterized.

Fig. 1

Current consensus of epigenetic gene regulation by noncoding RNAs. microRNAs is classified as one of small noncoding RNAs, and epigenetically function as negative regulator of protein coding genes by complementary binding to the target messengerRNAs (mRNAs). ncRNA, noncoding RNA.

MICRORNAS BIOGENESIS

miRNAs are evolutionarily conserved and are located either within the introns or exons of protein-coding genes (70%) or in intergenic regions (30%). Most intronic and exonic miRNAs are derived from their host genes, which suggests that they are transcribed concurrently with their host transcripts. Transcripts containing primary miRNAs (pri-miRNAs), which can vary from 200 nt to several kb in length, are capped with a specially modified nucleotide at the 5'-terminus and are polyadenylated with multiple adenosines at the 3'-end. Pri-miRNA is cleaved into precursor-miRNA (pre-miRNA: 60–70 nt in length) by the RNAse III enzyme (also known as Drosha). Pre-miRNA is exported from the nucleus into the cytoplasm by exportin-5. In the cytoplasm, pre-miRNA is cleaved by another RNase III enzyme, known as Dicer, into miRNA duplexes of approximately 19–22 nt in length. One miRNA duplex is then recruited into the RNA-induced silencing complex and functions to recognize complementary sites within the target messenger RNA (mRNA), thereby regulating translation through mRNA cleavage, degradation, or transcriptional repression [12].

MICRORNA PROFILING STUDIES IN BLADDER CANCER

miRNA expression profiling by array-based and polymerase chain reaction (PCR)-based methods is not commonly used because these methods only allow for identification of a limited number of known miRNAs. The rapid development of high-throughput, deep sequencing (DS) technology has provided novel information regarding the functions and features of miRNAs. Han et al. [13] first reported miRNA profiling in BC by using an Illumina Genome Analyzer IIX in 2011. Soon thereafter, five studies used DS technology to establish the miRNA profiles of clinical BC samples [1415161718]. To date, the largest DS study on BC is a comprehensive investigation by The Cancer Genome Atlas Research Network [18]. Tissues from 131 patients with high-grade MIBC were analyzed by RNA sequencing together with 118 blood samples from the same individuals and 23 adjacent histologically proven normal tissues. The aim of the project was to carry out genetic and epigenetic characterization of the molecular landscape of high-grade MIBC. In our studies, we conducted miRNA profiling by use of both PCRbased methods and DS technology [1719]. When comparing our 2 studies, the altered expression of miRNAs detected by DS technology was very similar to the results of PCRbased methods. In addition, a considerable number of novel miRNAs have been reported by some studies [131417]. Therefore, this analysis supports that DS technology is superior to previously used methods. Although PCR-based and DS profiles are not comparable from a quantitative point of view, sequencing approaches should provide substantial improvements to transcriptome analyses.

MOLECULAR TARGETS OF MICRORNAS IN BLADDER CANCER

1. Features of miRNA studies in BC

Growing evidence has demonstrated that aberrantly expressed miRNAs can act as oncogenic miRNAs (oncomiRNAs) or tumor-suppressive miRNAs (TS-miRNAs) in BC [1220]. Because miRNAs can construct miRNA-target gene networks that contribute to tumor initiation, survival, and invasion, many investigators have focused on the genes targeted by aberrantly expressed miRNAs in BC in comparison with normal bladder epithelium. In terms of miRNA analysis, luciferase-reporter assays are useful for confirming the direct binding of miRNAs to the specific 3'-untranslated region of the mRNA representing the target gene. Hence, we have summarized the miRNA studies in BC for which the target genes were validated by luciferase-reporter assays. To date, we have identified 78 studies that satisfied our criteria (Tables 1 and 2). As shown in Table 1, several TS-miRNAs, including miR-1, miR-16, miR-24, miR-101, miR-125b, miR-129, and miR-133a, have been shown to be located on different chromosomal regions but to have common sequences in their mature miRNAs. This suggests that the expression of these TS-miRNAs may be maintained by another genomic region, even though one region may be functionally disordered because of methylation, deletion, or mutation. This type of backup system is thought to have important roles in protecting normal cells from carcinogenesis.

Table 1

miRNAs and their target genes validated by lusiferase reporter assay

miRNAs	Stem-loop sequence	Locus	Clustered miRNAs	miRNA type	Target	Reference
miR-1	miR-1-1	20q13	ND	TS	LASP1	38
	miR-1-2	18q11	miR-133a-1		PNP	73
					PTMA	73
					SRSF9	26
					TAGLN2	74
miR-16	miR-16-1	13q14	miR-15a	TS	CCND1	75
	miR-16-2	3q25	miR-15b
miR-23b	miR-23b	9q22	miR-27b	TS	EGFR	24
			miR-3074		MET	24
			miR-24-1		ZEB1	40
miR-24	miR-24-1	9q22	miR-23b, miR-27b, miR-3074	TS	CARD10	76
	miR-24-2	19p13	miR-23a, miR-27a		FOXM1	32
miR-27a	miR-27a	19p13	miR-23a, miR-24-2	TS	RUNX1	77
					SLC7A11	78
miR-27b	miR-27b	9q22	miR-23b, miR-24-1, miR-3074	TS	DROSHA	79
					EGFR	24
					MET	24
miR-29c	miR-29c	1q32	miR-29b-2	TS	CDK6	80
miR-30a	miR-30a	6q13	ND	TS	NOTCH1	81
miR-34a	miR-34a	1p36	ND	TS	CD44	66
					HNF4G	82
					NOTCH1	83
miR-99a	miR-99a	21q21	let-7c	TS	FGFR3	46
miR-100	miR-100	11q24	ND	TS	MTOR	68
miR-101	miR-101-1	1p31	miR-3671	TS	COX2	84
	miR-101-2	9p24	ND		MET	85
					VEGFC	52
miR-124-3p	miR-124	20q13	ND	TS	ROCK1	86
					CDK4	87
miR-125b	miR-125b-1	11q24	ND	TS	E2F3	31
	miR-125b-2	21q21	ND		MMP13	88
					SPHK1	89
miR-128	miR-128-1	2q21	ND	TS	VEGFC	49
	miR-128-2	3p22	ND
miR-129	miR-129-1	7q32	ND	TS	GALNT1	25
	miR-129-2	11p11	ND		SOX4
miR-133a	miR-133a-1	18q11	miR-1-2	TS	EGFR	43
	miR-133a-2	20q13	ND		FSCN1	37
					GSTP1	90
					LASP1	38
					PNP	73
					PTMA	73
					TAGLN2	74
miR-133b	miR-133b	6p12	miR-206	TS	AKT1	47
					BCL2L2	47
					EGFR	43
miR-135a	miR-135a-1	3p21	ND	TS	FOXO1	91
	miR-135a-2	12q23	ND
miR-138	miR-138-1	3p21	ND	TS	ZEB2	41
	miR-138-2	16q13	ND
miR-143	miR-143	5q32	miR-145	TS	SERPIN1	22
					AKT	48
miR-144-5p/3p	miR-144	17q11	miR-451a, miR-451b, miR-4732	TS	CCNE1	28
					CCNE2
					CDC25A
					PKMYT1
miR-145	miR-145	5q32	miR-143	TS	CBFB	92
					CLINT1	92
					FSCN1	37
					ILK	48
					PAK1	93
					PPP3CA	92
					SERPIN1	22
					SOCS7	94
					IGF1R	44
miR-186	miR-186	1p31	ND	TS	HMGN5	95
miR-193a-3p	miR-193a	17q11	ND	TS	LOXL4	96
					PSEN1	52
					HOXC9	97
miR-195	miR-195	17p13	miR-497	TS	BIRC5	17
					CDC42	98
					CDK4	99
					GLUT3	100
					WNT7A	17
miR-200b	miR-200b	1p36	miR-200a, miR-429	TS	MMP16	39
miR-200c	miR-200c	12p13	miR-141	TS	BMI1	30
					E2F3
miR-203	miR-203	14q32	miR-203b	TS	BCL2L2	65
					BIRC5
miR-214	miR-214	1q24	miR-199a-2, miR-3120	TS	PDRG1	101
miR-218	miR-218-1	4p15	ND	TS	BMI1	67
	miR-218-2	5q34	ND		LASP1	38
miR-221	miR-221	Xp11	miR-222	TS	STMN1	102
miR-320a	miR-320a	8p21	ND	TS	ITGB3	103
miR-320c	miR-320c	18q11	ND	TS	CDK6	104
miR-449a	miR-449a	5q11.2	miR-449b, miR-449c	TS	CDC25A	105
miR-485-5p	miR-485	14q32	ND	TS	HMGA2	106
miR-490-5p	miR-490	7q33	ND	TS	FOS	107
miR-493	miR-493	14q32	miR-337, miR-665	TS	FZD4	51
					RHOC
miR-497	miR-497	17p13	miR-195	TS	BIRC5	17
					WNT7A
miR-574-3p	miR-574	4p14	ND	TS	MESDC1	108
miR-576-3p	miR-576	4q25	ND	TS	CCND1	109
miR-590-3p	miR-590	7q11	ND	TS	TFAM	110
miR-1182	miR-1182	1q42	ND	TS	TERT	111
miR-9	miR-9-1	1q22	ND	Onco	CBX7	112
	miR-9-2	5q14	ND
	miR-9-3	15q26	ND		CERS2	113
miR-10b	miR-10b	2q31	ND	Onco	HOXD10	70
					KLF4
miR-19a	miR-19a	13q31	miR-17, miR-18a, miR-20a, miR-19b-1, miR-92a-1	Onco	PTEN	114
miR-96	miR-96	7q32	miR-18, miR-183	Onco	CDKN1A	29
miR-150	miR-150	19q13	ND	Onco	PDCD4	115
miR-155	miR-155	21q21	ND	Onco	DMTF1	69
miR-182-5p	miR-182	7q32	ND	Onco	RECK	116
					SMAD4
miR-708	miR-708	11q14	ND	Onco	CASP2	27

miRNA, microRNA; TS, tumor suppressive miRNA; Onco, oncogenic miRNA; ND, not determined.

Table 2

Functional annotation of the target genes and the relative miRNAs

Annotation	Target	miRNA	miRNA type	Target validation in clinical BCs	Reference
Apoptosis	BCL2L2	miR-133b, miR-203	TS	yes	47,65
	BIRC5	miR-195, miR-203, miR-497	TS	yes	17,65
	BMI1	miR-200c, miR-218	TS	no	30,67
	CASP2	miR-708	Onco	yes	27
	SOCS7	miR-145	TS	no	94
	SOX4	miR-129	TS	yes	25
	SRSF9	miR-1	TS	yes	26
Cell cycle	CCND1	miR-16, miR-576-3p	TS	yes	75,109
	CCNE1	miR-144-5p/3p	TS	yes	28
	CCNE2	miR-144-5p/3p	TS	yes	28
	CDC25A	miR-144-5p/3p	TS	yes	28
	CDC25A	miR-449a	TS	yes	105
	CDK4	miR-124-3p	TS	no	87
	CDK4	miR-195	TS	yes	99
	CDK6	miR-29c, miR-320c	TS	yes	80,104
	CDK6	miR-449a	TS	yes	105
	CDKN1A	miR-96	Onco	yes	29
	E2F3	MiR-125b, miR-200c	TS	yes	30,31
	FOXM1	miR-24	TS	yes	32
	PKMYT1	miR-144-5p/3p	TS	yes	28
	STMN1	miR-221	TS	no	102
EMT	CARD10	miR-24	TS	no	76
	CBX7	miR-9	Onco	yes	112
	FSCN1	miR-133a	TS	yes	37
	FSCN1	miR-145	TS	yes	37
	HMGA2	miR-485-5p	TS	no	106
	HMGN5	miR-186	TS	yes	95
	LASP1	miR-1, miR-133a, miR-218	TS	yes	38
	LOXL4	miR-193a-3p	TS	no	96
	MESDC1	miR-574-3p	TS	no	108
	MMP13	MiR-125b	TS	no	88
	MMP16	miR-200b	TS	no	39
	ZEB1	miR-23b	TS	yes	40
	ZEB2	miR-138	TS	yes	41
Cytokaine-cytokaine receptor intaraction	EGFR	miR-23b, miR-27b, miR-133a, miR-133b	TS	yes	24,85
	IGF1R	miR-145	TS	no	44
	MET	miR-23b, miR-27b, miR-101	TS	yes	24,85
MAPK signaling	FGFR3	miR-99a, miR-100	TS	yes	46
	FOS	miR-490-5p	TS	yes	108
	RECK	miR-182-5p	Onco	yes	116
Notch signaling	NOTCH1	miR-30a, miR-34a	TS	yes	81,83
PI3K-Akt signaling	AKT1	miR-133b	TS	yes	47
	AKT1	miR-143	TS	no	48
	ILK	miR-145	TS	no	48
	MTOR	miR-100	TS	yes	68
	PTEN	miR-19a	Onco	no	114
Ras signaling	CDC42	miR-195	TS	yes	98
	PAK1	miR-145	TS	no	93
	RHOC	miR-493	TS	no	51
	ROCK1	miR-124-3p	TS	yes	86
VEGF signaling	VEGFC	miR-101	TS	yes	49,52
WNT signaling	FZD4	miR-493	TS	no	51
	PSEN1	miR-193a-3p	TS	no	52
	WNT7A	miR-195, miR-497	TS	yes	17
Metabolic	CERS2	miR-9	Onco	yes	113
	COX2	miR-101	TS	no	84
	GALNT1	miR-129	TS	yes	25
ECM-receptor	CD44	miR-34a	TS	yes	66
	ITGB3	miR-320a	TS	yes	103

miRNA, microRNA; TS, tumor suppressive miRNA; Onco, oncogenic miRNA; ND, not determined.

The miRNAs in Table 1 are listed in order of miRNA number, and the validated target genes are indicated. Interestingly, among the 54 miRNAs examined, 45 were downregulated, whereas 9 were upregulated. In our experience, we often encountered difficulties with the use of anti-miRNAs in in vitro studies; however, miRNA restoration systems using miRNA transfection are well established and commercially available. Accordingly, the latter system may be easier to use to examine the potential tumor-suppressive roles of the downregulated miRNAs.

miRNAs have been shown to regulate target gene expression. Several miRNAs and their targets have been identified in BC (Table 2). Among the 61 target genes examined, the expression levels in clinical BC specimens compared with the levels in normal bladder epithelium were validated for 40 genes. The downregulation of several miRNAs, including miR-1, miR-133a, miR-145, miR-195, and the miR-200 family, has been reported, and these miRNAs have been shown to be involved in crucial cellular processes, such as the epithelial-mesenchymal transition (EMT), cell cycle, apoptosis, and cancer signaling pathways. TS-miRNAs and their target genes were classified into functional annotations by using the Kyoto Encyclopedia of Genes and Genomes pathways (Table 2).

2. Clustered miRNAs

Several miRNAs are located in close proximity (within 10 kbp) in the same genomic region; this is called a miRNA cluster. Because of their simultaneous expression, common target genes of miRNAs within a cluster may be important for tumor biology. For example, miR-145 forms a cluster with miR-143 on chromosome 5q32, and these clustered miRNAs are regulated by a common promoter [21]. Research indicates that both miR-145 and miR-143 are frequently downregulated in a broad range of cancers, including BC. Villadsen et al. [22] suggested that SERPINE1 is a direct target of both miR-145 and miR-143; this was the first report of an miRNA cluster targeting the same mRNA in BC. Importantly, miR-145 has been implicated in the p53 regulatory network in human malignancies [23]. p53 transcriptionally induces the expression of miR-145 by interaction with a potential p53 response element in the miR-145 promoter; moreover, MYC is directly repressed by miR-145. Additionally, Chiyomaru et al. [24] found direct binding of the miR-23b/27b cluster with EGFR/MET, and Itesako et al. [17] found direct binding of the miR-195/497 cluster with BIRC5, which encodes survivin.

3. Cell cycle/apoptosis-related genes

Avoidance of apoptosis and cell cycle acceleration are required for cancer cells to escape cell death. BCL2L2, a member of the BCL2 family, can promote cell survival by inhibiting the intrinsic pathway of apoptosis. In contrast, BIRC5 (survivin) is a key member of the inhibitor of apoptosis protein family. Several TS-miRNAs induce apoptosis through targeting BCL2L2 (miR-133b, miR-203) and BIRC5 (miR-195, miR-203, miR-497). Upregulation of miR-129 may be involved in avoidance of apoptosis through direct targeting of SOX4 and GALNT1, which are involved in transcription and protein expression, respectively [25]. One important pathway is the sequential activation of caspases, which have a central role in the execution of cell apoptosis. miR-1 is downregulated in both clinical BC tissues and cultured BC cells and has been shown to induce apoptosis by increasing the activity of caspases 3 and 7 through direct targeting of the apoptosis inhibitor SRSF9 [26]. Additionally, CASP2 (caspase 2) is directly targeted by an onco-miRNA, miR-706 [27].

Several miRNAs have been shown to control the cell cycle through their target genes. Matsushita et al. [28] first reported that cell-cycle-related genes, such as CCNE1/2, CDC25A, and PKMYT1, are directly regulated by miR-144-5p. CDC25A is also regulated by miR-449a, and CDK4 and CDK6 are regulated by miR-124-3p, miR-195, miR-29c, miR-230c, and miR-449a, inducing G1-phase arrest. Interestingly, miR-96, which is upregulated in BC, directly binds to CDKN1A (p21), a well-known tumor-suppressive gene [29]. Moreover, miR-125b and miR-200c target the oncogene E2F3, which is critical for the G1/S transition and is overexpressed in most high-grade BCs [3031]. Inoguchi et al. [32] found that miR-24 regulates FOXM1, which is critical for mediating cell cycle progression proteins, such as Aurora B, cyclin B1, and CDC25B, and stimulates the expression of SKP2/CKS1, which is involved in the proteolysis of p27kip1 and in G1/S progression.

4. EMT-related genes as targets of miRNAs in BC

We also evaluated whether the expression of the different target genes could be validated in clinical BC samples (Table 2). The EMT is a key progression mechanism that is often activated during cancer invasion and metastasis and is characterized by loss of cell adhesion, repression of E cadherin expression, and increased cell mobility [33]. Members of the miR-200 family are well-known regulators of the EMT in human malignancies, including BC [34], and some investigators have reported the functions of these miRNAs in BC [3536].

FSCN1 and LASP1 are components of the filopodia and lamellipodia in various types of cells; both proteins are involved in mediating the dynamics of actin filaments and are activated during the EMT. miR-133a and miR-145 directly regulate FSCN1 [37], and the miR-1/133a cluster and miR-218 regulate LASP1 [38]. Restoration of the expression of these miRNAs accelerates cell migration and invasion activity in vitro [3738]. Matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endopeptidases that are capable of degrading various extracellular matrix proteins. MMPs are involved in the cleavage of cell surface receptors, the release of apoptotic ligands (such as the FAS ligand), and chemokine/cytokine inactivation. MMPs are also thought to facilitate tumor cell invasion and metastasis via the degradation of the extracellular matrix. Transforming growth factor beta1 reduces the expression level of miR-200b, which directly regulates MMP16. This then activates other MMPs (e.g., MMP-2 and MMP-9), growth factors, and receptors, thereby facilitating local cellular mechanisms that promote migration [39].

Loss of expression of miR-23b, miR-138, and miR-200c (an miR-200 family member) is associated with increased expression of ZEB1, ZEB2, and ERFFI1, which facilitate the progression of the EMT [364041]. ZEB1 and ZEB2 are known to negatively regulate E cadherin expression and are directly targeted by miR-23b and miR-138, whereas ERRFI-1, a regulator of EGFR, is directly targeted by members of the miR-200 family [36]. ZEB1 expression has been reported in 22% of clinical BC tissue specimens, but is absent from the bladder mucosa [42]. Expression of the miR-200 family has been shown to be repressed by hypermethylation of its promoter region, and loss of miR-200c expression is significantly associated with subsequent disease progression to MIBC and poor outcomes [35]. These studies implied that many EMT-related genes are activated through downregulation of several TS-miRNAs, and these phenomena may contribute to tumor progression of BC.

5. Others

Downregulated miRNAs have also been reported to target some genes related to cytokine–cytokine receptor interactions, including EGFR (miR-23b, miR-27b, miR-133a, miR-133b) [2443], IGF1R (miR-145) [44], and MET (miR-23b, miR-27b, miR-101) [2443]. Activation of these genes may affect downstream cancer pathways, such as mammalian target of rapamycin signaling, phosphoinositol 3-kinase (PI3K)-Akt signaling, and mitogen-activated protein kinase (MAPK) signaling. FGFR3, which belongs to the MAPK signaling pathway, is crucial for BC oncogenesis, and mutations in FGFR3 have been shown to accelerate proliferation in BC [45]. Downregulation of miR-99a and miR-100 has been shown to cause upregulation of FGFR3 expression before its mutation, which suggests that the acquisition of mutations may result from increased cell turnover [46]. The expression of AKT1 and ILK, which are involved in the AKT-PI3K pathway, was shown to be repressed by the miR-143/145 cluster, and proliferation is inhibited in cells transfected with these miRNAs [4748]. The vascular endothelial growth factor (VEGF) and WNT signaling pathways are also important for BC development, and VEGFC, FZD4, PSEN1, and WNT1 have been reported to be under the control of several TS-miRNAs [1749505152].

MICRORNAS AS TUMOR MARKERS IN BLADDER CANCER

1. miRNAs as diagnostic markers for BC detection

Many studies have investigated miRNA expression by quantitative real-time PCR of clinical BC specimens [20]. In this review, we focus on studies in which the investigators evaluated diagnostic accuracy by use of receiver-operator curve analyses of the sensitivity and specificity of specific miRNAs in comparison with normal bladder epithelium. We found 18 studies that satisfied our criteria (Table 3). In 2009, Ichimi et al. [19] first evaluated the expression of six miRNAs in tissues as diagnostic markers to distinguish BC (n=104) from non-BC (n=31).

Table 3

miRNAs as diagnostic markers

Sample	Study	Year	Calculator	BC/Cont. (n)	Sensitivity	Specificity	Reference
Tissue	Liu	2015	miR-141	30/30	78.2	51.6	139
			miR-200b		81.3	47
	Ichimi	2009	miR-30a-3p	104/31	94.3	76.7	19
			miR-125		89.4	77.4
			miR-133a		93.3	77.4
			miR-145		90.5	77.4
			miR-195		91.4	80
			miR-199a*		72.1	90.3
Urine	Long	2015	miR-26a/miR-93/miR-191/miR-940	85/45	70	84	56
	Wang	2015	miR-214	192/169	90.5	65.6	57
	Zhang	2014	miR-99a, miR-125b	50	86.7	81.1	58
	Zhou	2014	miR-106b	112/78	76.8	72.4	59
	Tolle	2013	miR-520e	36/19	70	63.2	117
			miR-618		70	68.4
			miR-1255b-5p		85	68.4
	Mengual	2013	miR-18a*/miR-25/miR-140-5p/miR-142-3p/miR-187/miR-204	151/126	84.8	86.5	61
	Shimizu	2013	miR-9-3/miR-124-2/miR-124-3/miR-137	86/20	81	89	55
	Yun	2012	miR-145	207/144	77.8	61.1	60
	Miah	2012	miR-15b/miR-135b/miR-1224-3p	121/53	94.1	51	118
	Dudziec	2011	miR-152/miR-328/miR-122-3p	68/53	81	75	62
			miR-212		40	66
			miR-328		59	91
			miR-1224-3p		84	58
	Yamada	2011	miR-96	100/74	71.8	89.2	54
			miR-183		74	77.3
	Hanke	2010	miR-126: miR-152 ratio	29/11	72	82	53
			miR-182: miR-152 ratio		55	82
Blood	Motawi	2016	miR-92a	70/62	97.1	76.7	119
			miR-100		90	66.7
			miR-143		78.6	93.3
	Fang	2015	miR-205	89/56	76.4	96.4	120
	Du	2015	miR-497/miR-663b	109/115	69.7	69.6	121
	Jiang	2015	miR-15b-5p/miR-27a-3p/miR-30a-5p/miR-148b-3p/miR-152/miR-3187-3p	110/110	90	90	63
	Tolle	2013	miR-26b-5p	38/20	65	94.1	117
			miR-144-5p		70	82.4
			miR-374-5p		60	94.1

miRNA, microRNA; BC, bladder cancer; Cont., control.

There is a pressing need for highly accurate, noninvasive tests for BC diagnosis. The standard procedures for BC diagnosis and the follow-up for patients depend on urinary cytology and cystoscopy. Urinary cytology is a reliable urine marker for BC diagnosis because of its high specificity (90%–95%). However, this method has low sensitivity (30%–40%), and patients are forced to undergo a painful cystoscopic procedure to confirm the diagnosis. A number of noninvasive urine tests, such as bladder tumor antigen and NMP22, have been developed; however, their diagnostic specificities are inferior to that of urinary cytology tests, and these methods have failed to meet clinical expectations. Therefore, quantitative changes in miRNAs in urine, blood, and tissues are the primary focus in the search for new markers. miRNAs are detectable in urine with pelleted or cell-free urine samples. Hanke et al. [53] evaluated miR-126 expression in whole urine samples and showed that this miRNA had a sensitivity of 72% and specificity of 82% when used as a diagnostic marker, even though the sample size in their study was small. Yamada et al. [54] also found that miR-96 detection in the urine including the pellet was a useful diagnostic marker, with a sensitivity of 71.8% and a specificity of 89.2%. Shimizu et al. [55] uniquely reported good sensitivity of 81% and good specificity of 89% by testing a methylation panel of miR-9-3/miR-124-2/miR-124-3/miR-137. In terms of miRNA detection in cell-free urine [5657585960], several studies have shown that this method provides moderate sensitivity and specificity for distinguishing BC from non-BC but that the results are inferior to those from studies using whole urine samples [5354556162]. In blood tests, Jiang et al. [63] showed dramatic results (sensitivity of 90% and specificity of 90%) using a panel study of miR-15b-5p/miR-27a-3p/miR-30a-5p/miR-148b-3p/miR-152/miR-3187-3p.

2. miRNAs for predicting prognosis in patients with BC

Many retrospective studies have suggested that the expression of specific miRNAs in BC specimens may be a good prognostic marker predicting overall survival, disease-free survival, recurrence-free survival, or progression-free survival, as outlined in Table 4. Moreover, several reports analyzing urine and blood samples have shown that miR-200, miR-214, and miR-3187-3p levels are good prognostic markers in the blood [57606364]. Surprisingly, miR-203, miR-214, miR-152, and miR-3187-3p were shown to be independent markers predicting overall survival, progression-free survival, or recurrence-free survival in multivariate studies [636465]. Unfortunately, no prospective studies have been performed to date. Thus, additional large, prospective studies are needed for the clinical application of miRNA assays for diagnostic and prognostic purposes in BC.

Table 4

miRNAs as prognostic markers

Sample	Study	Year	Calculator	BC (n)	Valuables	Multivariate	Reference
Tissue	Zhang	2016	miR-30a	50	OS, RFS	N/A	81
	Xu	2015	let-7c, miR-99a, miR-125b-1, miR-193a	202	OS	N/A	124
	Zhang	2015	miR-21	53	OS	N/A	122
	Wang	2015	miR-141	114	DSS	N/A	127
	Avgeris	2015	miR-143, miR-145	133	OS, PFS	N/A	125
	Zhang	2015	miR-203	108	OS, PFS	Independent	65
	Wu	2015	miR-424	124	OS, RFS	N/A	123
	Segersten	2014	miR-10a-5p	86	PFS	N/A	129
	Lin	2014	miR-26a	126	OS, DFS	N/A	126
	Zhang	2014	miR-222	97	OS	N/A	128
	Pignot	2013	miR-9, miR-182, miR-200b	166	OS, RFS	N/A	134
	Majid	2013	miR-23b	20	OS	N/A	40
	Dyrskjot	2009	miR-29c, miR-129, miR-133b	106	PFS	N/A	25
	Rosenberg	2013	miR-29c*	108	PFS	N/A	132
	Wang	2013	miR-31	126	OS, PFS	N/A	131
	Ratert	2013	miR-141, miR-205	40	OS	N/A	130
	Pignot	2012	miR-19a, miR-20a, miR-92a	71	OS	N/A	138
	Zaravinos	2012	miR-21	77	OS	N/A	136
	Wang	2012	miR-100	126	OS, PFS	N/A	133
	Hirata	2012	miR-182-5p	18	OS	N/A	116
	Puerta-Gil	2012	miR-222, miR-143	37	OS, DSS, RFS, PFS	N/A	137
	Majid	2012	miR-1280	21	OS	N/A	135
Urine	Dudziec	2011	miR-152 CpG, miR-1224 CpG	68	PFS	N/A	62
	Yun	2012	miR-200	138	RFS	N/A	60
	Wang	2015	miR-214	192	OS, RFS	N/A	57
	Kim	2013	miR-214	138	RFS	Independent	64
Blood	Jiang	2015	miR-152, miR-3187-3p	110	RFS	Independent	63
	Fang	2015	miR-205	89	DSS	N/A	120

miRNA, microRNA; BC, bladder cancer; OS, overall survival; DSS, disease specific survival; PFS, progression free survival; RFS, recurrence free survival; N/A, not applicable.

MICRORNAS AS A TREATMENT MODALITY IN BLADDER CANCER

Some TS-miRNAs have been transfected into BC cell lines by using plasmid/virus vectors, followed by subcutaneous injection of the transfected cells into nude mice [276667]. Significant inhibition of tumor growth [2767] and metastasis [66] was observed. Yu et al. [66] found that the CD44 gene was a direct target of miR-34a and that the pro-apoptotic role of miR-34a may be mediated primarily through regulation of CD44, which functions to promote metastasis and angiogenesis. Xu et al. [68] demonstrated that miR-100-transfected BC cells exhibited significant growth inhibition in an intravesical orthotopic BC model. In terms of onco-miRNAs, miR-10b- and miR-155-transfected BC xenografts exhibited significantly increased growth in comparison with controls [6970]. Inamoto et al. [71] tested the anticancer effects of intravesical injection of miR-145 by using cationic liposomes (Lipofectamine RNAiMAX, Thermo Fisher Scientific, Waltham, MA, USA) in an intravesical orthotopic BC model. They achieved adequate anticancer effects after intravesical administration of miR-145. These miRNAs are promising candidate miRNAs that may be useful for clinical applications in the future.

FUTURE PERSPECTIVES

Many studies have examined the roles of miRNAs in human malignancies, including BC. However, the majority of miRNA studies have focused on candidate gene approaches with limited whole-genome sequencing, and a detailed characterization of miRNA profiles based on the genomic features of BC has not been reported. Recently, DS technology has been applied for profiling of mRNA and miRNA in human cancers, including BC. However, DS technology has mainly focused on BC tissues without any particular selection according to clinical treatment. Additional DS analyses should be conducted using samples from patients who have undergone different treatments, e.g., chemotherapy regimens, radiation therapies, or immune-checkpoint drugs.

A growing body of evidence has shown that miRNAs have great potential for clinical use as new diagnostic biomarkers. However, detection of single miRNAs in urine samples will not be superior to current urinary cytology tests because of the relatively low diagnostic specificity of such analyses. Some recent studies have attempted to improve the diagnostic specificity of miRNA detection by using a panel of multiple miRNAs [55566163]. It will be important to select optimal combinations of miRNAs and to perform prospective translational studies in outpatients to validate such miRNA panels.

Moreover, recent studies have shown that some miRNAs control the activity of major cancer-related signaling molecules. Thus, identification of aberrant miRNA expression and oncogenic or tumor-suppressive molecular targets of miRNAs is necessary for the clinical development of novel cancer therapeutics. Because many TS-miRNAs and their target oncogenes are components of complex molecular networks, treatment with multiple miRNAs may provide stronger anticancer effects than treatment with a single miRNA. In an interesting study by Liu et al. [72], new vectors, termed "miRNA-mowers," that contained the entire sequence of the onco-miR-183-96-182 cluster were constructed. Transfection of the miRNA-mowers strongly inhibited cell growth and migration and induced apoptosis in vitro, suggesting the usefulness of targeting multiple onco-miRNAs. However, no studies have yet reported successful results for intravenous administration of TS-miRNAs in the induction of anticancer effects in vivo. The development of an adequate drug delivery system for TS-miRNAs is needed; otherwise, it will be difficult to determine the value of miRNAs as a therapeutic modality.