MicroRNA Signature in Renal Cell Carcinoma

Renal cell carcinoma (RCC) includes 2.2% of all diagnosed cancers and 1.8% of cancer-related mortalities. The available biomarkers or screening methods for RCC suffer from lack of sensitivity or high cost, necessitating identification of novel biomarkers that facilitate early diagnosis of this cancer especially in the susceptible individuals. MicroRNAs (miRNAs) have several advantageous properties that potentiate them as biomarkers for cancer detection. Expression profile of miRNAs has been assessed in biological samples from RCC patients. Circulatory or urinary levels of certain miRNAs have been proposed as markers for RCC diagnosis or follow-up. Moreover, expression profile of some miRNAs has been correlated with response to chemotherapy, immunotherapy or targeted therapeutic options such as sunitinib. In the current study, we summarize the results of studies that assessed the application of miRNAs as biomarkers, therapeutic targets or modulators of response to treatment modalities in RCC patients.


INTRODUCTION
Renal cell carcinoma (RCC) is the 15 th most frequent cancer, based on the statistics provided by GLOBOCAN (1). This kind of cancer includes 2.2% of all diagnosed cancers and 1.8% of cancerrelated mortalities (1). The incidence of this type of cancer is different in different regions. RCC is associated with numerous risk factors among them are smoking, obesity, and hypertension (2). The varied incident and mortality rates of RCC in different geographical regions necessitate enactment of regional screening programs and development of precise biomarkers (2). Among the screening methods for sporadic RCC, urine dipstick has yielded low level of accuracy impeding its clinical application (3). Moreover, none of the proposed serum and urine markers such as aquaporin 1, perilipin 2, and KIM1 had enough sensitivity or specificity to be applied in this regard (4). On the other hand, computed tomography and abdominal ultrasound suffer from high cost and low sensitivity for the identification of small tumors, respectively (2,3). Therefore, development of effective non-invasive screening methods for RCC is a necessity. Recent investigations have potentiated microRNAs (miRNAs) as screening tools for several kinds of human malignancies (5). These transcripts contribute in the pathogenesis of human disorders. In this review, we clarify the main points of studies in RCC to judge the potential of miRNAs as biomarkers or therapeutic targets in this malignant condition.

miRNA Biogenesis and Function
miRNAs have sizes about 23 nucleotides and are present in different species. By acting as antisense transcripts, miRNAs posttranscriptionally decrease expression of their targets. Although the regulatory effects of each miRNA on the expression of its target gene is not great, the resultant interactive network between miRNAs, target genes and downstream effectors plays crucial impacts on the regulation of cellular functions (6). The majorities of these transcripts are transcribed from DNA templates into primary miRNAs and undergo a number of steps to be processed into the precursor and mature miRNAs, respectively (7). Two kinds of RNase III molecules, i.e., Drosha and Dicer proteins participate in the miRNA processing in the nuclear and cytoplasmic cellular compartments, respectively (7). The critical function of miRNAs in gene expression modulation is additionally highlighted by the point that an individual gene is concurrently regulated by several miRNAs, and each miRNA can modulate expression of several targets which have sequence complementarity with its seed region (8). About one-third of human genome and virtually all essential cell processes are expected to be regulated by miRNAs (9,10). The role of miRNAs in the pathogenesis of human cancers has been vastly examined (11). These molecules have been reported to influence the main features of carcinogenic process such as sustained proliferative capacity, evasion from growth inhibitor signals, resistance to apoptosis, induction of invasive and metastatic programs, and enhancement of angiogenic processes (12). The importance of miRNAs in development of cancer has been firstly highlighted through the spotting miR-15a and miR-16-1 in a commonly deleted region in B-cell chronic lymphocytic leukemias (13). Subsequent investigations revealed other genomic alterations in a number miRNA coding genes in different cancers such as lung cancer (14), melanoma as well as ovarian and breast cancers (15). Moreover, well-known oncogenes such as c-Myc were shown to influence expression of oncogenic activates miRNAs including miR-17-92 (16) or inhibit expression of tumor suppressor miRNAs including miR-15a, miR-26, miR-29, miR-30, and let-7 (17). In RCC, quite a lot of investigation have measured expression profile of miRNAs in different biological samples to identify the pathogenic roles of these transcripts in the development of this type of cancer (18).

Dysregulated miRNAs in RCC
A number of studies have assessed differentially expressed miRNAs and their target genes in RCC samples and normal control. Using this approach, Li et al. have identified down-regulation of 521 genes and up-regulation of 473 genes in RCC samples. Proteinprotein interaction network showed RHCG, RALYL, SLC4A1, UMOD, and CA9 as nodes with high degrees of interactions. The differentially expressed genes were enriched in cytokine and cytokine receptor pathway (19). Such approaches are useful in identification of biomarkers and therapeutic targets for RCC. Other studies have reported dysregulation of a number of miRNAs in RCC samples. Figure 1 shows a number of dysregulated miRNAs in RCC and their interaction with the PTEN tumor suppressor.
The following sections describe the function of these miRNAs.
urogenital system (20). Wulfken et al. have investigated miRNA signature in both tissue and serum specimens of patients with RCC. They reported over-expression of 109 circulatory miRNAs in cancer patients; among them were 36 miRNAs that were upregulated in tissue samples as well. Additional verification steps indicated up-regulation of miR-1233 in another cohort of RCC patients. Notably, expression patterns of this miRNA in patients with angiomyolipoma or oncocytoma was similar with RCC patients (21). miR-301a is another up-regulated miRNA in RCC cell lines and clinical samples. Over-expression of this miRNA has been associated with advanced stage and poor survival of RCC patients. Mechanistically, miR-301a has been displayed to target PTEN tumor suppressor (22). Two other oncomiRs, namely, miR-22 and miR-193a-3p also suppress expression of PTEN in RCC cells (23,24). In addition, miR-1293 has been upregulated in RCC cells enhancing viability of these cells their migratory potential and invasiveness. These effects are mediated through suppression of Hydrocyanic Oxidase 2 (25). Table 1 gives a summary of the roles of up-regulated miRNAs in RCC. Notably, level of this miRNA was commonly decreased in urine specimens of patients as well (123). A comprehensive assessment of miRNA profile in plasma specimens of ccRCC patients and healthy subjects has revealed the correlation between circulating miRNA signature and ccRCC stage. miRNA profiles were remarkably different between stage III/IV sections and both controls and early stage samples. Plasma levels of miR-150 were considerably correlated with patients' survival (124). A large-scale detection of formerly unannotated miRNA sequences in human renal specimens has led to identification of several miRNAs being dysregulated in ccRCC tumors and linked with poor survival of patients (125). Finally, experiments in a transgenic model of Xp11 RCC have shown higher amounts of miR-204-5p in urinary exosomes compared with control animals. Expression of this miRNA was also elevated in primary RCC cell lines created from transgenic mice indicating its role as a diagnostic marker for Xp11 tRCC (126). Table 3 gives a brief record of studies which reported the diagnostic/prognostic role of miRNAs in RCC.

The Role of miRNAs in Determination of Response of RCC Patients to Treatment Modalities
Expression profile of a number of miRNAs correlates with response of RCC cells to chemotherapeutic agents. For instance, miR-381 has been shown to improve response of RCC cells to 5-fluorouracil through targeting WEE1 and enhancing activity of cyclindependent kinase 2 (128). Expression of miR-451 has been elevated in low multi-drug resistant (MDR);,;, cell line compared with the high MDR cell line. This miRNA has been shown to target ATF-2 and suppress its expression. Up-regulation of miR-451 has increased drug resistance, while its silencing improved response to chemotherapeutic agents (118). In the clinical settings, serum levels of miR-183 have been shown to predict response of RCC patients to cytotoxic effects of natural killer cells (129), implying the importance of miRNAs in immunotherapeutic options. A genome-wide miRNA profiling in RCC patients who received sunitinib showed lower levels of miR-141 in tumor samples of poor responders compared with good responders (81). Therefore, miRNAs modulate response of RCC patients to a wide range of treatment modalities. Table 4 summarizes the impact of miRNAs in resistance to therapeutic modalities in RCC.

DISCUSSION
The oncogenic function of numerous miRNAs has been proved in RCC cells These oncomiRs have been shown to enhance cell proliferation and invasive features of RCC cells whilst decreasing apoptosis Notably some tumor suppressor genes such PTEN APC and MEG3 have been identified as targets of oncomiRs such as miR-301a miR-193a-3p miR-22 miR-671-5p, and miR-7, indicating a possible mechanism for their participation in the pathogenesis of RCC. Instead, tumor suppressor miRNAs which are down-regulated in RCC cells have potential roles in the activation of apoptotic pathways and arrestment of cell cycle transition. A number of these miRNAs target EMT-associated genes such as ZEB1, Slug, HOTAIR, and HIF-1a. Thus, their down-regulation is associated with the enhancement of EMT program. miRNAs are regarded as potential markers of different malignancies including RCC. These transcripts regulate several cancer-related cellular functions such as apoptosis, survival, migration and angiogenesis. Therefore, several miRNAs have similar functions and expression profiles in diverse cancers.
Although aberrant expression of miRNAs in cancer patients is a useful tool for follow-up of patients, identification of tissuespecific pattern of their expression is necessary to differentiate between different cancers originating from a certain body system. In spite of extensive efforts for biomarker discovery, there is no consensus on miRNA panels that are specific for a certain type of cancer. A previous study has reported up-regulation of miR-28, miR-185, miR-27, and let-7f-2 in RCC samples, whereas   expression of a different set of miRNAs including miR-223, miR-26b, miR-221, and miR-103-1was increased in bladder cancer samples. Based on these results, authors suggested the potential of miRNAs in differentiating between these two types of cancers (20). However, others have reported over-expression of bladder cancer-related miRNAs such as miR-223 and miR-221 in RCC samples (33,130) casting doubt on the possibility of identification of tissue-specific miRNA signature in different cancers. Studies which appraised the biomarker role of miRNAs in RCC suffer from small sample size, inclusion of samples from diverse clinical stages and histologic subclasses as well as benign kidney lesions and validation in independent samples. Possibly, the most important limitation of miRNAs as diagnostic markers is their inability for differentiation between malignancies with diverse origins. Based on this limitation, they cannot be used for primary diagnosis of cancer but for patients' follow-up. Another possible application of miRNAs in the RCC patients rises from their importance in the determination of patients' response to chemotherapy. Therefore, a prior identification of miRNA profile in the biopsy samples might facilitate selection of the most appropriate therapeutic regimen in a personalized manner. Moreover, targeted suppression of certain miRNAs is a possible modality to enhance response of patients to chemotherapy. miR-21 represents a promising candidate in this regard, since it has been shown to be overexpressed in RCC samples in independent studies and its silencing has enhance response to multiple anti-cancer drugs such as paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib. Yet, miRNA-based therapies face a number of challenges such as design of specific formulations to avoid off-target effects and low efficacy of delivery methods (131). Comparison of miRNA levels in serum and tissue samples of RCC patients and healthy subjects has led to identification of several dysregulated miRNAs in serum samples. Yet, only a fraction of these miRNAs have been dysregulated in tissue samples, implying that a minor portion of circulating miRNAs have been originated from the tumor tissues (21). Therefore, future studies are needed to explore the source of circulating miRNAs in RCC patients. Based on the results of recent investigations, both serum and urine samples of patients with RCC might be used as sources for discovery of miRNA levels, facilitating conduction of non-invasive methods for RCC diagnosis. miRNA signature can be used for classification of RCC subtypes. The miRNA-based classification system developed by Youssef et al. could discriminate different subtypes of RCC such as clear cell, papillary, oncocytoma, and chromophobe RCC with sensitivity values between 97% and 100% (132). Moreover, miR-15a has been shown to have distinct expression pattern between RCC and oncocytoma being up-regulated in the former, yet down-regulated in the latter. Expression of this miRNA was similarly up-regulated in chromophobe carcinoma, while in the papillary RCC samples miR-15a expression was not such overexpressed. Over-expression of miR-15a was also detectable in urine samples of RCC patients. However, miR-15a was almost miR-124 -FZD5, P-gp Wnt signaling pathway miR-124 promotes cell apoptosis, and inhibits chemoresistance.
ANNTs, adjacent non-tumoral tissues. Patients with higher levels of miR-210 and miR-1233 display a significantly lower cancer-specific survival.
-- ( 33) 148 ccRCC tissue samples along with 60 ANTTs Patients with high miR-122 levels display a significantly lower metastasis-free survival rates than those with low miR-122 levels.
High miR-122 level is a poor prognostic factor for metastasis High miR-122 is an independent prognostic factor from gender, age, BMI, overall TNM, tumor size, grade and staging.  Overexpression of miR-10a-5p and miR-10b-5p and downregulation of miR-223-3p were significantly correlated with survival.
miR-301a is an independent prognostic marker for RCC patients. Taken together, miRNAs participate in the pathogenesis of RCC and response of patients to diverse therapeutic modalities. Moreover, as they are traceable in circulation and urine samples of patients, they can be used as biomarkers for this kind of cancer. However, at the present time, there is no miRNA that can be widely applied as biomarker or treatment target in the clinical setting. This is partly because of the heterogeneous pattern of expression of miRNAs in RCC samples and circulation of patients. This research filed lacks comprehensive assessment of miRNA profiles in large cohorts of RCC patients. Therefore, future studies with these features are expected to facilitate design of suitable diagnostic panels containing miRNAs.

AUTHOR CONTRIBUTIONS
MT and SG-F wrote the draft and revised it. ZS-F and WB designed the tables and study, and performed the data collection. All authors contributed to the article and approved the submitted version.