Identification of biological targets of therapeutic intervention for clear cell renal cell carcinoma based on bioinformatics approach

Renal cell carcinoma (RCC) represents the most lethal genitourinary malignancy and about 90 % of all kidney tumors are RCC [1]. Clear cell RCC (ccRCC) is the most common subtype of RCC, accounting for about 75 % of cases [2]. Because of being generally asymptomatic, one-third of ccRCC are already metastatic at initial diagnosis, leading to a high mortality rate up to 95 % [3]. However, the benefit of surgical, chemotherapeutic and radiological approaches for ccRCC is limited [3, 4]. Thus, it is of great importance to gain a better understanding of the pathogenesis of ccRCC, which may facilitate the development of effective biomarkers and novel, targeted therapeutic strategies.

Major efforts have been carried out to explore the underlying etiology and molecular mechanisms of ccRCC. Cigarette smoking, obesity, hypertension, and acquired cystic kidney disease were shown to be associated with renal cancer [5]. There is ample evidence indicating that the most frequent event in ccRCC is inactivation or mutations in the von-Hippel Lindau (VHL) tumor suppressor gene, which induces the expression of the hypoxia inducible factors (HIF)1α and HIF2α [6, 7]. Two of the other most commonly mutated genes in ccRCC are PBRM1 (about 40–50 %) and BAP1 (10–15 %) [8]. In addition, extracellular signal-regulated kinase 5 (ERK5) is demonstrated to mediate angiogenesis, proliferation, and tumor aggressiveness in ccRCC [9]. By contrast, microRNAs (miRNAs), a group of small non-coding RNAs (21–25 nucleotides) that regulate expression of target genes at the post-transcriptional level, are crucial for gene-regulatory networks which play an important role in different biological processes, including metabolic disorder, tissue injury, and oxidative stress [10, 11]. Aberrant alterations in miRNA expression have been shown to be related to human malignancies [12, 13]. Numerous miRNAs have also been identified in ccRCC in which some were demonstrated to function as oncogenes, while others were identified to play tumor suppressor roles [14, 15]. For instance, Mathew et al. have demonstrated that the suppression of miR-30c-2-3p as well as miR-30a-3p increases HIF2α levels in ccRCC, promoting angiogenesis, cellular proliferation, and tumor growth [16]. In addition, Liu et al. have suggested miR-23b* was up-regulated in renal cancer as an important regulator of proline oxidase [17]. Additionally, a number of array platform-based studies recently indicate that a considerable number of miRNAs are dysregulated in ccRCC [18]. Although the results of previous studies may not be consistent, all the data indicate that dysregulated miRNAs may play pivotal roles in the pathogenesis of ccRCC. However, the present knowledge is still poorly understood.

In the current study, we employed the bioinformatics methods to identify the differentially expressed genes (DEGs) and differentially expressed miRNAs between ccRCC tumors and corresponding non-tumor samples. Target genes of differentially expressed miRNAs were screened. Then, functional enrichment analysis of DEGs was performed, followed by protein–protein interaction (PPI) network construction and sub-module analysis. Finally, an integrated miRNA-DEG network was constructed. We intended to give a systematic perspective on understanding the molecular mechanism and to investigate more potential therapeutic targets for ccRCC.

ccRCC is the most common histological subtype of RCC that occurs in adults and associated with worse prognosis [33]. In this study, we applied bioinformatics method to predict the potential miRNA targets for the treatment of ccRCC progression. Our results suggested that 1758 up- and 2465 down-regulated DEGs were screened out in ccRCC samples. Moreover, a total of 15 up- and 12 down-regulated differentially expressed miRNAs were identified. The up-regulated DEGs were enriched in significant pathways such as CAMs and focal adhesion. Besides, the down-regulated DEGs were significantly associated with oxidative phosphorylation, and TCA cycle. Several significant differentially expressed miRNAs were identified and miRNA-200 family was found to be the most significant.

miRNA-200 family includes miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429, and miRNA-141 [34]. In the present work, miR-200a, miR-200b, miR-200c and miR-429 were identified from the network of DEGs and their related miRNAs. Additionally, these miRNAs were down-regulated in ccRCC. As previously reported, the members of the miRNA-200 family (especially miR-200c and miR-141) play an outstanding role as metastasis suppressor genes via inhibiting the expression of zinc finger E-box binding homeobox 1 (ZEB1) [35, 36]. Under-expression of miRNA-200 family members is correlated with renal cancer [37]. Moreover, the elevation of collagens and fibronectin in obstructed kidneys can be repressed by the injection of miR-200b [38]. Previous reports exhibited that the increased level of type V collagen has been detected in human breast cancer and in mouse skin tumors [39, 40]. In the current study, we found that under-expression of miR-200c targeted and up-regulated the level of collagen, type V, alpha 2 (COL5A2) and COL5A3. Besides, we found that the up-regulated DEGs were significantly associated with CAMs and focal adhesion (Table 4). Amounting evidence has demonstrated that collagens and fibronectin contributes to cell adhesion dynamics and cell migration which are significantly concerned with tumor metastasis [41, 42]. Taken together, we infer that miRNA-200 family may regulate several genes such as COL5A2 and play a critical role in the progression in ccRCC through participating in cell adhesion and migration. The members of the miRNA-200 family, especially miRNA-200c, may constitute novel therapeutic targets in ccRCC and further experimental verifications are needed.

Out of the highly up-regulated miRNAs, miRNA-15a was a member of the miRNA-15 precursor family including miRNA-15a, miRNA-15b, miRNA-16-1, miRNA-16-2, miRNA-195 and miRNA-497. Metabolic activities for energy in normal cells depend on mitochondrial oxidative phosphorylation (OXPHOS) primarily, but OXPHOS capacity is decreased in various cancer cells [43]. The process of OXPHOS needs oxygen. Oxygen deficiency leads to the inhibition of OXPHOS, mainly mediated by the HIF-1 [44]. HIF is a master regulator of RCC metabolism [45]. Moreover, the miRNA-15a can regulate oxygen consumption and adenosine triphosphate (ATP) production via targeting uncoupling-protein 2 [46]. The low ATP synthase often observed in ccRCC [47]. In the present study, the up-regulated miRNA-15a was identified to regulate the ATPase, H+ transporting, lysosomal 21 kDa, V0 subunit b (ATP6V0B) gene which was significantly enriched in the pathway OXPHOS. Moreover, pathway enrichment analysis of down-regulated genes showed these genes were significantly linked with oxidative phosphorylation (Table 5). Hence, we infer that miRNA-15a may play a crucial role in the pathogenesis of ccRCC via being involved in oxidative phosphorylation. miRNA-15a may provide a novel therapeutic target in ccRCC and further experiments are needed to verify this finding.

Another up-regulated miRNA in human cancers is miRNA-155, which is described as an oncogene [48]. Previous studies demonstrated that miRNA-155 was up-regulated in various kinds of human malignancy, including breast cancer, non-small cell lung cancer and lymphomas [49‐51]. Recent studies have also exhibited the up-regulation of miRNA-155 levels in renal cancers [52, 53]. In accordance with the previous studies, our results found that miRNA-155 was up-regulated in RCC samples. Moreover, the inhibition of miRNA-155 expression induced apoptosis, suppressed proliferation and migration in renal cancer cells [54]. Collectively, our results further confirmed that therapy targeting miRNA-155 inhibition may be an effective approach for ccRCC treatment.

However, our study has several limitations. For instance, analysis of the other data in similar topic with larger samples may be beneficial to cross-check of the results in our study. Besides, there was a lack of experimental verifications. We intend to perform further experimental verifications in our future studies using different methods as the other study performed [14, 19], such as immunohistochemistry, and quantitative RT-PCR.

In summary, the identified DEGs and their related miRNAs, especially miRNA-200c and its target genes COL5A2 as well as COL5A3, miRNA-15a and its target genes ATP6V0B and miRNA-155 may play key roles in the progression of ccRCC, and these may be useful biomarkers for predicting tumor metastasis and therapeutic targets for the treatment of ccRCC. However, further experiments are needed to validate the effects and mechanisms of miRNA-200c, miRNA-15a and miRNA-155 in ccRCC.