A Mini Review on Epigenetics MiRNAs and their Influence on Bladder Cancer Development

Bladder cancer is the 10th most common cancer worldwide, with significant morbidity and mortality. Its early diagnosis is crucial and there is an urgent need for the development of non-invasive and specific biomarkers to detect bladder cancers at an early stage. Epigenetic alterations are innovative cancer biomarkers that show great potential for assay development to assist in patient management because of their stability and accessibility in body fluids. Recent studies demonstrated that microRNAs (miRNAs) are molecules involved in the epigenetic events emerging as diagnostic biomarkers and either their downregulation or upregulation occurs through epigenetic changes. Urological neoplasms (prostate, bladder, kidney), lung, breast and colorectal cancers are the most common and despite major advances in their characterization, this has not yet translated into biomarkers suitable for clinical practice. However, large multicenter validation studies are required to foster translation to the clinics. Herein we review the most promising epigenetic, diagnostic and predictive biomarkers in different types of biological sample related to patients harbouring bladder cancer.


Mini Review
Among all malignancies, bladder cancer (BCa) deserves particular attention, since is the most common malignancy of the urinary tract in the world. Approximately 70-75% are non-muscle invasive bladder cancer (NMIBC) with an incidence of 430.000 cases newly diagnosed every year causing over 165.000 cancerrelated deaths mortality [1]. It affects the superficial mucosa and submucosa with reduced tendency to cancer progression and high long-term survival. In 25-30% of cases, the disease invades the muscle layer of the bladder wall and it is defined muscleinvasive bladder cancer (MIBC) with a high risk of mortality [1].
Current diagnostic and prognostic strategies, such as tumor grade, stage, size and number of foci, are partially useful for clinical decision-making, in fact, the early diagnosis, disease recurrence and progression are the difficulties for clinicians when caring for patients with BCa. Cystoscopy and voided urine cytology (VUC) are specific diagnostic tools for BCa [2]. Although considered the gold standard, cystoscopy is both invasive and expensive for the patient and can miss certain lesions, in particular small areas of carcinoma in situ (CIS). VUC has a median sensitivity of only 35%, and a median specificity of 94% [3] although it is useful for detecting high-grade tumors and CIS. However, because of its low sensitivity in lowgrade cancers, it has limited clinical relevance. The limitations of VUC and the invasiveness of cystoscopy have generated interest in other non-invasive tools.

Molecular Markers in BCa
As in other forms of cancer, BCa also shows chromosomal anomalies. With fluorescence in situ hybridization (FISH) techniques, chromosomal anomalies can be detected in exfoliated bladder cells. Several studies reported low sensitivity of FISH to detect low-grade (36%-57%) and low-stage (62%-65%) tumors, while has high sensitivity for both high-grade and high-stage tumors (83%-97%) [4,5]. The detection of CIS is close to 100% and its hight specificity (89%-96%) is comparable to cytology [6]. An important multicentre study conducted on 1182 subjects affected by painless hematuria define the ability of immunocytology to predict the likelihood of BCa [7]. Immunocytology is based on the visualization of tumor-associated antigens in BCa using monoclonal antibodies and results showed that sensitivity, specificity, positive and negative predictive value of immunocytology for predicting BCa were 82.4%, 86.6%, 61.6%, and 95.0%, respectively. These data confirm immunocytology as a strong BCa predictor, outperforming cytology and helping in the clinical decision-making process.
From voided urine, Eissa and colleagues, analyzed relative telomerase activity by telomeric repeat amplification protocol (TRAP assay), human telomerase RNA (hTR-mRNA) by RT-PCR and human telomerase reverse transcriptase (hTERT) by RT-PCR. Overall the sensitivity of human telomerase reverse transcriptase for detecting BCa was the highest compared to that of human telomerase RNA, relative telomerase activity and urine cytology (96%, 92%, 75% and 75%, respectively). Specificity was 96%, 89%, 92% and 94%, respectively. hTERT followed by hTR provided sensitivity of 100% and specificity of 85% and thus an improvement in diagnosis of BCa [8]. In a systematic review, Glas showed that telomerase had the best sensitivity (75%) compared to the other markers, including cytology [9]. Among all candidate biomarkers for BCa, cytokeratin 20 (CK20), a low molecular-weight protein, measured on tissue from human primary and metastatic carcinomas after surgical removal, is considered a marker of urothelial differentiation [10] and several studies explored its role at the mRNA and protein level [11,12]. A recent study, highlighted the use of CK20 and p53 immunohistochemistry in bladder specimens with borderline or suspicious features for urothelial CIS and correlate them with subsequent or prior cancer diagnoses [13] Significant difference in CK20 expression was found between low and high grade urothelial carcinomas; a diffuse positive expression in 68.8% and 40.6% of high and low grade tumors, was shown, respectively. Strong positive expression of p53 was seen in 72.9% of cases of high grade, while only 36.2% cases of low-grade tumors revealed strong expression. Moreover, they verified the combined expression of P53 and CK20 in 40% of high grade and 22% of lowgrade carcinomas [13].

Implication of MiRNAs During Carcinogenesis
The recent literature highlights the importance of specific molecules that regulate protein-coding genes post transcriptionally in higher eukaryotes. Of particular interest in this field of molecular research are miRNAs [14], a class of non-coding RNAs of 19 to 24 nucleotides that regulate gene expression through posttranscriptional pathways, RNA interference and gene silencing [15]. Similar to epigenetic mechanisms, miRNAs appear to be important in the onset and development of several diseases. In humans, miRNAs involvement in cancer is spurring the interest of scientist [16], since they contain a great amount of information about the regulatory mechanisms of the cells. In attempts to classify various forms of cancer with expression profiles, miRNAs have been shown to harbor more information about disease states than mRNA profiles do [17]. MiRNAs are transcribed using RNA polymerase II into an up to 3000 nucleotide long primary miRNA (pri-miRNA), which is processed by Drosha to the precursor miRNA (pre-miRNA); a single pri-miRNA may contain multiple miRNA precursors [18]. The RNAse III enzyme Dicer cuts the pre-miRNAs proliferation [19]. The different expression of these molecules is easily measured, and the miRNA expression profiles were shown to be altered in tumor cells compared with normal tissues [20].
Recently, miRNAs have been identified in exosomes where their biogenesis, release and uptake may involve the endosomal-sorting complex required for transport (ESCRT). This machinery is made up of cytosolic protein complexes, known as ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, together with a number of accessory proteins, playing a pivotal role in cellular processes including multivesicular body biogenesis of exosome, cellular abscission and viral budding [21]. Exosomes are 40-100 nm nano-sized vesicles that are released from many cell types into the extracellular space.
Such vesicles are widely distributed in various body fluids [22].   As a result, they found that members of the let-7 miRNA family are abundant in exosomes derived from AZ-P7a, but are less abundant in exosomes derived from other cancer cells [28]. Moreover, some reports have shown that exosomal miRNA expression levels are altered under different physiological conditions. The serum level of exosome containing miR-21 was lower in healthy donors than patients with glioblastoma [29]. Levels of let-7f, miR-20b, and miR-30e-3p were lower in vesicles from plasma of non-small-cell lung carcinoma patients than normal controls. Different levels of eight exosomal miRNAs, including miR-21 and miR141, were also found between benign tumors and ovarian cancers [30].
According to these previous studies, a class of miRNAs are preferentially sorted into exosomes, such as members of the miR-320 family that are widely distributed in exosomes derived from normal tissue and tumors [31]. In addition, some miRNAs, miR-451 for example, are highly expressed in exosomes derived from normal cells, such as the HMC-1 cell line and the HEK293T cell line [32].
In summary, miRNAs present specific sequences that may guide their incorporation into exosomes, whereas some enzymes or other proteins may control exosomal miRNAs sorting as well, in a miRNA sequence-independent fashion. The evidences highlight the role of miRNAs for the diagnosis and staging of BCa that could provide great improvements in clinical decision-making.

MiRNAs in BCa
The complex regulatory mechanisms of miRNAs that cause the degradation of the target mRNA or the inhibition of translation, define the role of miRNAs as oncogenes or tumor suppressor genes. In this contest, downregulated miRNAs that target tumor suppressor genes are considered to be onco-miRNAs, and upregulated miRNAs that target oncogenes are considered to be tumor-suppressing miRNAs [33]. Similar to other malignancies, BCa is characterized by an alteration of the molecular profile caused by the accumulation of genetic and epigenetic modification.
Several reports compared the presence of circulating miRNAs resulted from bladder cells undergoing necrosis and apoptosis with modifications of the normal bladder phenotype [34]. For these reasons, miRNAs related to bladder carcinogenesis were gradually explored, raising the interest of several research groups in using miRNA as tools for diagnosis, prediction of tumor progression and BCa metastasis [35]. A recent work performed a global miRNA profiling of 392 serum samples of BCa patients with 100 non-cancer samples and 480 samples of other types of cancer as controls by a miRNA microarray [36]. They identified 7 miRNA candidates (miR-6087, miR-6724-5p, miR-3960, miR-1343-5p, miR-1185-1-3p, miR-6831-5p and miR-4695-5p) that clearly discriminated BCa from non-cancer controls and other types of cancer, regardless of cancer stage and grade, enabling the specific and early detection of BCa with high accuracy. Among these, miR-6087 showed high diagnostic performance for BCa screening [36]. MiR-141 and miR-205 are poor prognostic biomarkers of overall survival in BCa [37], whereas severely decrease of miR-29c * is able to discriminated 50% of NMIBC in advanced cancer [38]. The expression profiles of miRNAs by either microarray or deep sequencing technologies, have been documented in numerous investigations confirming their putative role as tumor marker for BCa diagnosis [39,40].

MiRNAs in Several Body Fluids Related to BCa
Advanced technology uses liquid biopsy for the analyses of circulating tumor cells (CTCs), exosomes, and circulating miRNAs in patients' blood and urine as samples instead of primary BCa tissue [47]. Several cell-free miRNAs, such as miR-497 and miR-214 were detected by liquid biopsy in plasma and urine of BCa patients, respectively [48,49]. Although this application could be promising for the discovery of novel circulating biomarkers, liquid biopsy in BCa treatment is yet to be explored and, at the moment, is used only Recent studies demonstrated that miRNAs are abundantly present in body fluid including serum, plasma, cerebrospinal fluid, saliva and urine and their expression is correlated with several diseases, including cancer [50]. Cells undergoing necrosis and apoptosis are the major source of circulating miRNAs. Tumor cells actively secrete cellular elements that can be detected in body fluids as either free circulating miRNAs or bound to ribonucleoprotein complexes and encapsulated in extracellular vesicles such as exosomes [51]. MiRNAs in exosomes may support tumor angiogenesis, invasion and metastasis through bone marrow-derived cells mobilization [52]. This encapsulated transport, complexed with proteins or lipids, protects miRNAs from degradation and is stable in body fluids. Thank to miRNAs capacity to be easily screened and precisely quantified by a variety of standard methods like qRT-PCR, in situ hybridization, enzymatic luminescence miRNA assay, microarray or next-generation sequencing [53], they can be clinically applied to detect tumors at an early stage. The stability, the frequency, the reversibility and the accessibility in body fluids of miRNAs as well as the resistance to various storage conditions and stability up to 24 h storage, make miRNAs optimal biomarker candidates of neoplastic diseases [54,55]. Even though total circulating miRNA levels are increased in cancer patients [56], the fraction of cancer-specific miRNAs is low and needs to be quantified compare to the background of "normal" miRNAs, thereby pointing out the need for sensitive and specific detection methods.  MiR-4454 and miR-21-5p were both detectable in FFPE-tumor tissue, urine exosomes and WBCs, while let-7a-5p, let-7b-5p, miR-451a and miR-16 were abundant in FFPE-tumor tissue, WBCs and plasma [58]. Moreover, an evaluation of the useful non-invasive biomarkers of low-grade tumors (Ta), was examined in matched bio-specimens from Ta patients only, between FFPE-tumor tissue and urine exosomes. The highest abundance of miR-4454, miR-720/3007a and miR-21 were found in both bio-specimen sources.
Interestingly, while correlations in the cellular expression of miR-125b, miR-100, and let-7a are mirrored in the EVs, tRNA-derived miRNA levels are found only in the EVs. These findings suggest the possible use of tRNA-derived small RNA fragments in combination with known miRNA signatures of tumors to distinguish circulating tumor-derived EVs from physiological EVs derived from other cell sources.
An important miRNA profiling investigation was conducted on 23-paired samples, normal and tumoral bladder tissue, by Braicu and colleagues [62]. They identified 18 downregulated and 187 upregulated miRNAs, by next-generation sequencing investigation, focalizing on a systematic identification of altered miRNAs and genes mutated involved in BCa tumorigenesis and progression. Among all these miRNAs, relevant findings were obtained concerning miR-21. As a miRNA commonly upregulated in human cancers, in vitro models demonstrated that miR-21 is able to regulate cell proliferation and migration via its cross talk with phosphatase and tensin homolog (PTEN) and tumor protein 53 (TP53), one of the most important pathways related to bladder carcinogenesis [63]. Moreover, a recent study confirmed the altered expression level for miR-21, miR-205 and miR-200c in BCa analyzing data generated by pairing the tissue to plasma samples [64]. Studies on chondrogenic differentiation of murine mesenchymal stem cells (MSCs) demonstrated that miR-145 is a negative regulator through directly targeting SRY-Box transcription factor 9 (SOX9), a key transcription factor for chondrogenesis [65]. Remarkably, SOX9 was significantly upregulated in tissues of patients with either renal cell carcinoma or BCa and correlated to the advanced pathological grade and clinical stage of patients [66].
Although there are no studies that confirm the correlation between miR-145 and proteins involved in cell differentiation such as SOX9 in BCa, Hashimoto and coworkers confirmed that miR-145 and miR-133 directly control fascin actin-bundling protein 1 (FSCN1), an actin-binding protein required for the formation of actin-based cell-surface protrusions and cytoplasmic bundles of microfilaments in BCa patients [67]. In summary, miR-145 and miR-133a might function as tumor suppressors through direct repression of FSCN1 in BCa.
The need to discriminate patients with BCa from those with benign diseases such as glomerulonephritis, benign prostate hyperplasia, infection, or urinary calculi lead Piao and colleagues to perform a multicenter study using 543 urine samples obtained from the National Biobank of Korea, including 326 BCa, 174 hematuria and 43 pyuria without cancer [68]. In this study, the expression ratio between urinary cell-free miRNAs, miR-6124 and miR-4511, was found to be significantly higher in patients with BCa than in those with hematuria and pyuria. Although these miRNAs ranged a sensitivity and specificity of 78.5% and 74.1%, respectively, were not sensitive enough to replace cystoscopy; in fact, avoiding