Key miRNAs and target genes played roles in the development of clear cell renal cell carcinoma

In order to investigate the functional information of target genes in the miRNA-target regulatory network, Database for Annotation, Visualization, and Integrated Discovery (DAVID) was applied to address it by web-based analysis modules, including Gene Ontology (GO) functional categories [22] and Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathway [23].

2.5 Survivability analysis of differentially expressed miRNAs and target genes

The online software SurvExpress (http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp) is an online biomarker validation tool for cancer gene expression data using survival analysis. SurvMicro (http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp) is an assessment of miRNA-based prognostic signatures for cancer clinical outcomes by multivariate survival analysis. In order to analyze the survivability of miRNAs and target genes in the miRNA-target regulatory network, the TCGA dataset was applied to analyze the survivability of selected miRNAs and genes in CCRCC patients by using SurvMivro and SurvExpress. In this study, we performed the SurvMivro/SurvExpress workflow as follows: (1) Input miRNA/gene list and select dataset; (2) Specify analysis, stratification and outputs; (3) Cox fitting, risk estimation and output writing; (4) Visualize plots and tables. The detailed processes are as follows: In the first Input web page, we paste the list of miRNA/gene and choose the dataset from available datasets; SurvExpress validates and searches the miRNA/gene and dataset to show the Analysis web page where we select the censored outcome and visualize the results. Only those miRNAs and genes with the survival curves not overlapping at one or more points were associated with the survival time of CCRCC patients.

3. Results

4. Discussion

CCRCC is the most common type of RCC, which affects the health of patients. Complementation of multidimensional omics data can remarkably increase the value of the study results and highlight the prominent associations between gene expression and cancer [24]. In this study, we integrated miRNA profiles and mRNA profiles data of CCRCC to find the value miRNAs and genes which were associated with the development of CCRCC. Integration of miRNA profiles led to 52 differentially expressed miRNAs and 2361 differentially expressed genes in CCRCC compared with normal tissue. According to miRNA-mRNA expression correlation analysis and miRNA target prediction, we obtained 89 miRNA-mRNA pairs under the thresholds of p_value 0.05 and r_value 0.3. The results suggested that these differentially expressed miRNAs and target genes may be associated with carcinogenesis of patients with CCRCC.

Hsa-mir-215 is involved in cancer cellular proliferation and migration, and can induce cell-cycle arrest by increasing the expression of p21 [25]. It is found that hsa-mir-215 is decreased in metastatic RCC samples compared to primary RCC tissues [12]. It is found that AK1 is significantly expressed in renal cells [26]. In this study, we found that hsa-mir-215 was up-regulated in the tumor tissues of CCRCC. One of target genes of hsa-mir-215, adenylate kinase 1 (AK1), was down-regulated in CCRCC. It is suggested that epigenetic modification regulation between hsa-mir-215 and AK1 was correlated with CCRCC progression. In addition, AK1 were closely associated with the survival time of CCRCC.

It is proposed that hsa-mir-199a-1 may target cancer-specific lncRNAs by miRNA response elements [27]. CDK18 belongs to the cell cycle-related CDK subfamilies, which play various functional roles by regulating different cell cycle progress [28]. Increased phosphorylation of CDK18 is found in the database of vasopressin regulation of kidney [29]. It is a putative up-regulated target gene of hsa-mir-101 in RCC specimens [30]. Herein, we first found that the expression of hsa-mir-199a-1 was down-regulated in the tumor tissues of CCRCC. Additionally, cyclin dependent kinase 18 (CDK18) was up-regulated and targeted by hsa-mir-199a-1. This indicated that the cell cycle-related gene CDK18 under the regulation of hsa-mir-199a-1 may serve as a biomarker for the early stages of CCRCC tumor development.

Lu et al. found that the expression of hsa-mir-204 was higher in human kidney compared with other organs tested [31]. It has been demonstrated that hsa-mir-204 is down-regulated and is a potential survival molecular biomarker of CCRCC [32, 33]. Moreover, the higher tumor grade of CCRCC is associated with a concomitant decrease in hsa-mir-204 [16]. LRG1 has been reported to play roles in cell proliferation, cell migration, cell apoptosis, immune response and neovascularization [34, 35, 36, 37]. It is worth mentioning that the missense mutations (L279F) in LRG1 gene is detected in the chromophobe RCC tissues [38]. In this study, we found that hsa-mir-204 was down-regulated. Furthermore, leucine rich alpha-2-glycoprotein 1 (LRG1), one of target genes of hsa-mir-204, was up-regulated in the tumor tissues of CCRCC. Our results provided additional evidence for their important roles of hsa-mir-204 and LRG1 in CCRCC.

It is reported that hsa-mir-501-5p is down-regulated and involved in the focal adhesion and extracellular matrix pathways in the papillary RCC [39]. Vila et al. found that the amount of GAPDH was increased in tumor tissues of CCRCC patients [40]. Scavenger receptor class B member 1 (SCARB1) has been suggested implicated in the development of CCRCC [41]. RNA-seq analysis showed that the expression of SCARB1 was up-regulated in CCRCC [42]. In addition, the mutation (rs4765623) in the SCARB1 gene has been found associated with CCRCC [43]. Banerjee et al. found that Heme oxygenase 1 (HMOX1, also called HO-1) was often over expressed in RCC, which protected cancer cells from apoptosis induced by chemo- therapeutic drug [44]. It is proposed that HMOX1 expression may be a useful biomarker for drugs response prediction for patients with metastatic CCRCC [45]. Our study found that hsa-mir-501 was down-regulated in CCRCC, which had a potential interaction with the up-regulation of GAPDH, SCARB1 and HMOX1. Our results suggested that hsa-mir-501 and target genes including GAPDH, SCARB1 and HMOX1 were correlated with CCRCC progression.

The expression of hsa-mir-2355 is up-regulated in lung adenocarcinoma compared with adjacent normal tissues [46]. In addition, hsa-mir-2355-5p has been detected in breast cancer when compared to normal tissue [47]. The expression of succinate-CoA ligase GDP-forming beta subunit (SUCLG2) is detected in the chromophobe RCC [48]. There is a significant decreased expression of SUCLG2 in CCRCC tissues compared with normal kidney tissue [49]. Herein, we found that hsa-mir-2355 was up-regulated in CCRCC and had a potential interaction with the down-regulation of SUCLG2, which indicated the important roles in the process of CCRCC.

Down-regulated expression of hsa-mir-200a is found in metastatic CCRCC compared to benign kidney tissues [50]. Additionally, the expression of hsa-mir-200a-3p in plasma is related to overall survival of metastatic kidney cancer [51]. It has been found that protocadherin gamma subfamily C, 3 (PCDHGC3) is expressed in RCC [52]. Protein S (PROS1) is up-regulated and involved in organismal and immune system of CCRCC [53]. In addition, genetic mutation of PROS1 (c.1294C $>$ T; p.R432W) is found in tuberous sclerosis complex-associated papillary RCC [54]. In the present study, we found that hsa-mir-200a was down-regulated, which was significantly inversely correlated with PCDHGC3 and PROS1. It is noted that PCDHGC3 was significantly associated with survival time of CCRCC patients. Our results provided additional evidence for their important roles of hsa-mir-200a and target genes including PCDHGC3 and PROS1 in CCRCC.

Hsa-mir-30c-2 is decreased in metastatic CCRCC compared to benign kidney tissue [50]. It is found that solute carrier family 9 member A9 (SLC9A9) is involved in chromosomal rearrangements in RCC [55]. Herein, we found that the expression of hsa-mir-30c-2 was decreased and inversely correlated with SLC9A9. In a word, the expression changes of SLC9A9 under the regulation of hsa-mir-30c-2 illuminated the mechanism of carcinogenesis which may play a crucial role in the progress of CCRCC.

Down-regulated hsa-mir-429 can significantly inhibit cancer cell proliferation, suggesting that hsa-mir-429 was a candidate tumor suppressive miRNA in RCC [56]. It is reported that hsa-mir-429 was down-regulated in CCRCC [32]. It is reported that egl-9 family hypoxia inducible factor 3 (EGLN3, also called PHD3) is important for the hypoxic cell cycle to proceed over G1/S checkpoint by reducing the stability of p27 in CCRCC cells [57]. Microarray analysis showed that EGLN3 was significantly up-regulated in CCRCC compared with normal tissue [58]. Furthermore, it has been demonstrated that high EGLN3 expression serves as the marker of poor prognosis in CCRCC patients [59]. In this study, we found that hsa-mir-429 was down-regulated, and the target gene, EGLN3, was up-regulated in CCRCC. Additionally, EGLN3 was significantly enriched in the renal cell carcinoma signal pathway. Our results suggested that epigenetic modification of EGLN3 in terms of hsa-mir-429 regulation was linked to CCRCC progression.

5. Conclusions

In summary, integrated analysis of the TCGA data led to a pool of differentially expressed miRNAs and DEGs. The epigenetic modifications between miRNAs (such as hsa-mir-215, hsa-mir-199a-1, hsa-mir-204, hsa-mir-501, hsa-mir-2355, hsa-mir-200a, hsa-mir-30c-2 and hsa-mir-429) and target genes including AK1, CDK18, LRG1, GAPDH, SCARB1, HMOX1, SUCLG2, SLC25A15, TOM1L2, PCDHGC3, PROS1, SLC9A9 and EGLN3 may be implicated in the growth of CCRCC. The epigenetic alterations of genes hold promise for becoming biomarkers for the prognosis of CCRCC. However, there is a limitation to our study. The sample size in the normal controls was small in the TCGA dataset. Larger numbers of normal controls that match the solid tumor are needed for further research.