A total of 50 ccRCC tissues and their matched normal adjacent tissues (>2 cm from the edge of the cancer tissue) were obtained from Fuzhou General Hospital (Fuzhou, Fujian) from November 2013 to November 2015. The separation and use of human tissues was approved by the Human Research Ethics Review Committee of Fuzhou General Hospital (approval no. 2013-017). Patients ranged in age from 28–77 years with the mean age of 55.5 years. All patients provided written informed consent. These patients were staged according to the Tumor-Node-Metastasis classification system of malignant tumors (7th) (11). Table I presents the patient's details.

A498 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China); CAKI-1 cells were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China). A498 cells are Von Hippel-Lindau tumor suppressor (VHL) mutant cells and CAKI cells are VHL wild-type cells (12). The cell lines used were all identified using short tandem repeat markers. A498 and CAKI-1 cells were maintained at 37°C and 5% CO₂ in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc.), and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.).

Analysis of gene expression omnibus (GEO) and the cancer genome atlas (TCGA) data of ccRCC. Oncomine (http://www.oncomine.org) was used to select highly expressed genes in Beroukhim Renal_GSE14994 (13), Grumz Renal_GSE6344 (14), Jones Renal_GSE15641 (15) and Lenburg Renal_GSE781 (16). Gene expression microarray datasets (GSE53757) (17) were analyzed and normalized by GEODiver (http://www.geodiver.co.uk). The primary dataset was downloaded from the NCBI GEO database (http://ncbi.nlm.nig.gov/geo) (18). Kidney Renal Clear Cell Carcinoma (KIRC) mRNA expression and clinical data was downloaded from TCGA (http://cancergenome.nig.gov) (19). The transcriptome profiling data files for analysis were normalized and transferred into a txt file using a Perl script. Subsequently, the package edgeR of Bioconductor was used in RStudio (version 3.5.0; http://www.rstudio.com/) to screen out the differentially-expressed genes (DEGs) with a fold change >2, P< 0.01 and a false discovery rate value <0.01, which were considered statistically significant (20). Relative gene expression of BHLHE41 was reflected by the intensity of the probe signal and plotted using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Kaplan-Meier survival curves were generated by Oncolnc (http://www.oncolnc.org/) for patients with ccRCC entered in TCGA database.

Methylation data (Illumina Infinium Human Methylation 450 K) (21), which included 325 ccRCC tumor samples and 160 normal samples, were downloaded from TCGA data portal in April 2018. Samples with expression and methylation data were screened. Individual gene methylation was extracted from the fasta file using a custom Perl script. The correlation between DNA methylation/loci methylation level and BHLHE41 gene expression was investigated with Spearman's test.

Total RNA Kit II Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) was used to extract RNA from tissues. Subsequently, 3 µg total RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). SYBR-Green select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to qPCR. cDNA was subjected to RT-qPCR with BHLHE41 and β-actin primers. BHLHE41 upstream primer, 5′-AAGGAGCATGAAACGAGACGA-3′, and downstream primer, 5′-CTCGGTTAAGGCGGTTAAAGC-3′. β-actin upstream primer, 5′-TGACGTGGACATCCGCAAAG-3′, and downstream primer, 5′-CTGGAAGGTGGACAGCGAGG-3′. PCR was conducted for 40 cycles with 20 pmol primers under the following conditions: 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec. Relative fold changes in mRNA expression were calculated by normalization to β-actin mRNA using the formula 2^−∆∆Cq (22).

Tissue samples, and A498 and CAKI-1 cell extracts were prepared by a Protein Extraction reagent (cat. no. P0013; Beyotime Institute of Biotechnology, Haimen, China). A Bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to quantify total protein. A total of 15 µg protein from each sample were separated on 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P PVDF Membrane; EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk at room temperature for 1 h, and then incubated with primary antibodies (BHLHE41, p-AKT, AKT, p-p70S6K, p70S6K, β-catenin, E-cadherin and ACTIN) overnight at 4°C. Subsequently, membranes were incubated with HRP-conjugated secondary IgG antibodies (anti-rabbit IgG, cat. no. 7074; and anti-mouse IgG, cat. no. 7076) (both 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 h. Proteins were detected using a SuperSignal West Pico chemiluminescent kit (Pierce; Thermo Fisher Scientific, Inc.). BHLHE41 antibody (cat. no. ab190093; 1:1,000) was purchased from Abcam (Cambridge, UK). phospho (p)-AKT (cat. no. 9271; 1:1,000), AKT (cat. no. 9272; 1:1,000), p-p70S6K (cat. no. 9234; 1:1,000), p70S6K (cat. no. 2708; 1:1,000), β-catenin (cat. no. 8480; 1:1,000) and E-cadherin (cat. no. 3195; 1:1,000) antibodies were purchased from Cell Signaling Technology Inc. β-actin antibody (cat. no. A2228; 1:5,000) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

BHLHE41 short hairpin RNA (shRNA; pLKO.1-BHLHE41-shRNA, TRCN0000016946) and non-targeting control SHCOO2 (shRNA-NC) vector were purchased from Sigma-Aldrich (Merck KGaA). For virus packaging, 3 µg non-targeting sequence control or BHLHE41 shRNA constructs were co-transfected with 1 µg pMD2.G and 2 µg psPax2 into 293T cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). A498 and CAKI-1 cells were infected with virus media. The media was changed after 48 h and RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin containing 1 µg/ml puromycin was added at 37°C for 10 days. Subsequently, the stably transfected cells were obtained. Inhibition efficiency of gene expression detected by RT-qPCR (data not shown) and verified with a western blot assay as previously described.

Selected transgenic A498 and CAKI-1 cell proliferation was measured using a WST-1 assay kit (Roche Diagnostics GmbH, Mannheim, Germany). At 24, 48, 72 and 96 h after cell plating at 37°C, 10% WST-1 solution was added to adherent cells, and then incubated at 37°C for 45 min. The coloration of the solution was measured at 450 nm using a microplate reader.

Selected transgenic A498 and CAKI-1 cells were seeded in a 24-well plate at 5×10³ cells/cm², and then cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C for 1 day, the cells were pulsed with 10 µM BrdU at 37°C for 1 h and fixed in 4% polyparaformaldehyde at room temperature for 15 min. Fixed cells were labeled with anti-BrdU antibody (cat. no. 5292; 1:100; Cell Signaling Technology, Inc.) at 4°C overnight, and then washed 3 times. Subsequently, the cells were treated with anti-mouse Alexa-488 (cat. no. 715-545-450; 1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 37°C for 30 min, and counterstained with DAPI (Sigma-Aldrich; Merck KGaA) at room temperature for 5 min. Photomicrographs were captured using an epifluorescence microscope (X81; Olympus Corporation, Tokyo, Japan; ×100).

Selected transgenic A498 and CAKI-1 cells were harvested for migration assays in 24-well plates with 8-µm pore size Transwell microporous membranes (EMD Millipore). Subsequently, 200 µl of the A498 and CAKI-1 cell suspension (containing 3×104 cells) was seeded in the upper chamber with serum-free RPMI-1640 medium, and the lower chamber was supplemented with 10% fetal bovine serum with 500 µl RPMI-1640 medium. After incubating for 36 h at 37°C in a 5% CO₂ atmosphere, the cells were washed with PBS 3 times and fixed in 4% paraformaldehyde at room temperature for 15 min. The cells on the upper surface of the membrane were then removed with a cotton swab, and the migrating cells on the bottom surface were stained with 0.5% crystal violet at room temperature for 4 h. The number of cells in 5 random fields was counted under an epifluorescence microscope (X81; Olympus Corporation; ×100) and the mean was recorded.

Data were collected from at least three technical replicates and expressed as the mean ± standard deviation. Statistical analyses of transcriptome profiling data results were performed in RStudio (version 3.5.0) software. Statistical differences between the groups were analyzed with the unpaired Student's t-test or one-way analysis of variance with the Bonferroni correction for multiple comparisons. All the data were graphically displayed using GraphPad Prism 6 software. P<0.05 was considered to indicate a statistically significant difference. Kaplan-Meier analysis of overall survival for patient's estimates based on all available data and compared with the log-rank test.

To search for ccRCC oncogenes, data mining was performed on the Oncomine database and 4 datasets were selected, which were analyzed using Affymetrix genome array (Human Genome U133A Array) (the detection method of the reference data). BHLHE41 was determined to be one of the highly DEGs (Fig. 1A). Fig. 1B depicts that BHLHE41 was the only overlapping gene among all of the 4 datasets. BHLHE41 is highly expressed in human ccRCC samples, compared with adjacent normal renal tissues (Fig. 1C) (Beroukhim Renal non-hereditary and hereditary ccRCC, n=59, fold change=19.414, P<0.001; Grumz Renal ccRCC, n=10, fold change=1.47, P<0.001; Jones Renal ccRCC, n=32, fold change=1.91, P<0.001; and Lenburg Renal ccRCC, n=12, fold change=1.32, P<0.001).

Subsequently, analysis of BHLHE41 expression from a large cohort ccRCC dataset (GSE53757, 72 pairs, HG-U133 Plus_2 array) was performed. BHLHE41 is one of the DEGs between ccRCC and their adjacent tissues (Fig. 2A and B). It was enhanced significantly in ccRCC tissues (P<0.0001), as demonstrated by paired Student's t-test analysis (Fig. 2C).

To further investigate the role of BHLHE41 in ccRCC, the expression of BHLHE41 was analyzed using TCGA's ccRCC (KIRC) RNA-seq data (21). The analysis results demonstrated that BHLHE41 was overexpressed in tumor tissues (P<0.0001; Fig. 3A). However, there were no significant differences among the various pathological grades (Fig. 3B) and high expression of BHLHE41 was not significantly associated with the overall survival rate in patients with ccRCC (Fig. 3C).

A total of 50 pairs of pathology confirmed and surgically removed ccRCC tissues, and their adjacent tissues, were collected at the Fuzhou General Hospital. The RT-qPCR data demonstrated that BHLHE41 was highly expressed in 94% of tumor tissues (Fig. 4A). Paired Student's t-test analysis revealed that BHLHE41 mRNA levels were significantly elevated in ccRCC tissues (P<0.0001; Fig. 4B). Subsequently, 5 pairs of samples were detected by western blot analysis. Fig. 4C indicates that the BHLHE41 protein level was increased in tumor tissues. For the samples collected, information on pathological Fuhrman grades (23), with the grades primarily being G1 and G2, was obtained, but there was no patient survival information. Therefore, an association between BHLHE41 expression and tumors was produced, but its association with tumor grade and patient survival was not analyzed.

BHLHE41 was stably knocked down in A498 and CAKI-1 cells using a BHLHE41 shRNA (shRNA-BHLHE41) or a scrambled control (shRNA-NC) to evaluate their proliferation and migration. Using WST-1 assays, it was observed that the knockdown of BHLHE41 in A498 and CAKI-1 cells significantly suppressed cell proliferation at 48, 72 and 96 h detection time points (Fig. 5A and B). The BrdU assay demonstrated that BHLHE41-knockdown cells proliferation rate was significantly reduced in both cell lines (Fig. 5C and D). BHLHE41-knockdown cell colonies were reduced, compared with shRNA-NC cells, in the ccRCC cell lines (Fig. 5E and F). Subsequently, a Transwell chamber assay was used to assess the effect of BHLHE41 on cell migration. Cell migration was significantly decreased in BHLHE41-knockdown A498 and CAKI-1 cells, compared with their control cells (Fig. 5G). These data indicate that BHLHE41 acts as a novel tumor-promoting molecule regulating ccRCC growth.

Since mechanistic target of rapamycin kinase (mTOR), AKT and epithelial-mesenchymal transition (EMT) signaling pathways are common tumorigenic pathways (24,25), to further determine the mechanism by which BHLHE41 drives the ccRCC tumorigenesis, western blot analyses was performed with a number of marker genes of mTOR, AKT and EMT signaling pathways. p70S6K activity was significantly reduced in BHLHE41 knockdown A498 and CAKI-1 cells. p-AKT levels were incomparable between the BHLHE41 knockdown and control cells. BHLHE41-knockdown cells and shRNA-NC cells exhibited relative high expression levels of E-cadherin protein in A498 and CAKI-1. There was no detected change in the amount of β-catenin (Fig. 6). These data indicated that the p-p70S6K and E-cadherin expression levels, which are involved in the mTOR and EMT pathways (26,27) respectively, were coordinated by BHLHE41.

The expression of BHLHE41 has been reported to be regulated by HIF-1α (9). To further investigate potential mechanisms underlying the transcriptional enrichment of BHLHE41, DNA methylation levels and their association between copy number changes were analyzed in TCGA ccRCC samples in the present study. The analysis data demonstrated reduced DNA methylation of BHLHE41 in tumor samples (Fig. 7A; P<0.0001). Potential correlation between methylation and BHLHE41 gene expression was statistically significant (cor=−0.644, P=9.115×10⁻⁴²; Fig. 7B). These results demonstrated that methylation changes are associated with BHLHE41 expression.

A total of 17 probes of BHLHE41 were included in further analysis, and 5 loci were determined to be significantly reduced in tumor samples (Table II). It was observed that the correlations between expression and methylation of 3 loci (cg13515269, cg16047471 and cg25629905) within the 3′UTR region were statistically significant (cor=−0.638 and P=7.069 ×10⁻⁴¹; cor=−0.429 an P=6.481×10⁻¹⁷; and cor=−0.541 and P=1.417×10⁻²⁷, respectively; Fig. 7C). These data indicated that increased BHLHE41 expression in ccRCC samples is associated with methylation level in the 3′UTR region.