The human renal cancer cell lines (786-Oand ACHN) and an immortalized proximal tubule epithelial cell line (HK-2) were obtained from American Type Culture Collection (Manassas, VA, USA). The human renal cancer cell line OS-RC-2 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The 786-O and OS-RC-2 cell lines were maintained in RPMI-1640 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), the ACHN and HK-2 cell lines were maintained in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences). The medium was supplemented with 10% fetal bovine serum (FBS; Shanghai ExCell Biology, Inc., Shanghai, China). All cells were cultured in a humidified atmosphere at 37°C in 5% CO₂ (17).

Small interfering RNA (siRNA) for CREB (siCREB; 5′-GUCUCCACAAGUCCAAACATT-3′; antisense, 5′-UGUUUGGACUUGUGGAGACTT-3′) and siRNA for negative control (siNC; sense, 5′-UCCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). A total of 5×10⁵ cells were seeded in 6-well plates for 24 h at 37°C and transfected with specific siCREB (100 pmol) or control siNC (100 pmol) by using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA, according to the manufacturer's protocol (22).

Cells were added to a 24 well-plate at a concentration of 0.7×10⁵ cells/well in 1 ml 10% FBS-containing medium. Cells were cultured for 24 h and reached 70% confluence at 37°C in 5% CO₂. Linear scratches were made with a micropipette tip across the diameter of the well and dislodged cells were rinsed with PBS. Cell culture medium was replaced with fresh, serum-free medium. Images of the scratch were acquired as baseline and images of the same location were obtained after 24 h. Experiments were performed in quadruplicate (23).

The cell invasion assay was performed using BD Matrigel 24-well 8 µm invasion chambers with filters coated with extracellular matrix on the upper surface (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, 100 µl serum-free medium containing 1×10⁴ cells from each subgroup were added to the upper chamber. The 600 µl medium (HyClone; GE Healthcare Life Sciences) containing 20% FBS (Shanghai ExCell Biology, Inc.) was added to the lower chamber as a chemoattractant. Cells were allowed to invade for 24 h at 37°C in 5% CO₂. The cells in the upper chamber were removed using a cotton swab. Cells that had migrated to the bottom of the membrane were fixed and stained 30 min with 0.1% crystal violet staining solution at room temperature and were photographed from 5 different microscopic fields (Motic AE31; Motic China Group Co., Ltd., Xiamen, China). Crystal violet was removed by addition of 33% acetic acid at 200 µl/well to the 96-well plates and absorbance was measured at 570 nm using a SpectraMax Plus384; Molecular Devices LLC (Sunnyvale, CA, USA). Experiments were performed in triplicate (23).

For western blot analysis, 50 µg of each sample were processed as described (24). CREB (catalog no. 9197), pCREB (Ser133; catalog no. 9198), N-cadherin (catalog no. 14215), E-cadherin (catalog no. 3195), β-actin (catalog no. 8457) and GAPDH (catalog no. 5174) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). MMP-2 (catalog no. ab7033), MMP-9 (catalog no. ab137651), vimentin (catalog no. ab8978) and fibronectin (catalog no. ab2413) antibodies were obtained from Abcam (Cambridge, UK). All primary antibodies were diluted at 1:1,000 and were used according the manufacturer's protocol. Goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP were used (dilution 1;5,000; catalog no BA1054/BA1050; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and detected by chemiluminescence. Densitometric analysis was performed by Tanon GIS version 4.1.2 software (Tanon Science and Technology Co., Ltd., Shanghai, China).

ChIP assays were performed using a SimpleChIP^® Enzymatic Chromatin IP kit (catalog no. 9002; Cell Signaling Technology, Inc.) according to the manufacturer's protocol. IgG antibody was included in the ChIP assay kit. pCREB (Ser133) antibody (dilution 1:50; catalog no. 9198) was obtained from Cell Signaling Technology, Inc. Quantification of immunoprecipitated DNA was performed using quantitative polymerase chain reaction (qPCR) with LightCycler 480 SYBR-Green I Master (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol. CREB binds to a target sequence termed the CRE-site and this serves as a model for the research of CREB function, and the CREB-binding protein (CRE-BP) site is the transcription factor CRE-BP binding site. The −449 to −442 bp (CRE site) fibronectin promoter sequence from the transcription start site had the following primers: 5′-CCGAAAAAAAGTTGTCTTGCCC-3′ (forward); and 5′-CAGCCGACCGCGCGCCGATTGG-3′ (reverse). The −731 to −722 bp (CRE-BP site) vimentin promoter sequence from the transcription start site had the following primers: 5′-TATTGCCGCCAAAGATTCTG-3′ (forward); and 5′-TACCCTGGTGGAAGTCATTAAAG-3′ (reverse). ChIP results were calculated and presented as a percentage relative to the input DNA by the ∆∆Cq method (25,26).

Using the standard TRIzol protocol (Invitrogen; Thermo Fisher Scientific, Inc.), total RNA was isolated and extracted from cells. The amounts of total RNA were quantified using spectrophotometric measurements. Using a RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, total RNA was reversed transcribed into cDNA. qPCR was performed using the LightCycler 480 SYBR-Green I Master (Roche Diagnostics). Each standard qPCR reaction contained the following; Reverse transcriptase product (2 µl); 0.5 µl of each primer (final concentration, 0.1 mM); 12.5 µl SYBR-Green PCR Master Mix™ (Roche Diagnostics). The thermocycling conditions included an initial 15 min holding period at 95°C, followed by a PCR program repeated for 50 cycles: 15 sec at 94°C, 57 sec at 30°C and 30 sec at 70°C. The following primers were used: MMP-2, 5′-TTGACGGTAAGGACGGACTC-3′ (forward) and 5′-ACTTGCAGTACTCCCCATCG-3′ (reverse); MMP-9, 5′-TTGACAGCGACAAGAAGTGG-3′ (forward) and 5′-CCCTCAGTGAAGCGGTACAT-3′ (reverse); GAPDH, 5′-AAGCCTGCCGGTGACTAAC-3′ (forward) and 5′-GCATCACCCGGAGGAGAAAT-3′ (reverse). GAPDH was used as a normalization and other gene expression levels were analyzed using the ∆∆Cq method (25). All samples were performed in triplicate and, for each reaction, negative controls without reverse transcriptase or RNA were performed (12,27).

All experiments were repeated 3 times. Using SPSS, version 18.0 (SPSS, Inc., Chicago, IL, USA), results are presented as the mean ± standard deviation. One-way analysis of variance and Fisher's least significant difference tests were performed to evaluate the differences between groups. P<0.05 was considered to indicate a statistically significant difference.

In the present study, to investigate, in multiple human RCC cell lines, the functional role of the upregulation of CREB, siCREB was used to knockdown the expression of CREB in 786-O, OS-RC-2 and ACHN RCC cells, and normal HK-2 cells. To detect the effect of siCREB, the expression of CREB was investigated by western blotting. The results demonstrated that the protein expression of CREB and pCREB were significantly downregulated compared with cells transfected with siNC and control, following cell transfection with siCREB (Fig. 1). In addition, cell migration and invasion ability was analyzed using wound healing and cell invasion assays. Transfection with siCREB significantly blocked migration (P<0.01; Fig. 2) and invasion (P<0.01; Fig. 3) in 786-O and OS-RC-2 cells compared with control, however, significantly increased migration (P<0.05; Fig. 2) and invasion (P<0.01; Fig. 3) in ACHN cells compared with control, and demonstrated no significant effects in HK-2 cells (Figs. 2 and 3). These results indicate that CREB may contribute to the migration and invasion phenotype of RCC cells, and not HK-2 cells, however, results were not consistent between the3 RCC cell lines.

MMPs may disrupt the extracellular matrix to promote cancer cell mobility, which eventually causes metastasis (28,29). Among MMPs, MMP-2 and MMP-9 are vital enzymes involved in the degradation of gelatin, collagen and laminin (30). The expression of numerous genes is activated by CREB (31). Therefore, the present study aimed to investigate whether CREB regulates RCC cell migration and invasion ability by affecting MMP-2/9 expression. As observed in the results of the wound healing and cell invasion assay, 786-O and OS-RC-2 cells, following siCREB transfection, expressed significantly lower levels of MMP-2/9 protein (P<0.01; Fig. 4A) and mRNA (P<0.01; Fig. 4B) compared with the siNC group, however, the opposite was observed in ACHN cells where protein and mRNA levels of MMP-2/9 were significantly increased compared with the siNC group (P<0.01; Fig. 4). Combined, the results demonstrate that CREB regulated the migration and invasion of the 3 types of RCC cells, however, the effects of CREB knockdown were different between the 3 RCC cell lines.

The EMT is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. A previous report demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion (32). Thus, in order to further investigate the association between CREB and EMT, markers associated with EMT were assessed using western blot analysis in 3 types of human RCC cell lines. The results demonstrated that protein levels of N-cadherin and fibronectin were significantly reduced (P<0.01; Fig. 5), and E-cadherin protein levels were significantly increased (P<0.01; Fig. 5) in siCREB-transfected 786-O and OS-RC-2 cells compared with the siNC group, however, the opposite was observed in ACHN cells, which corresponds with the results obtained in wound healing and invasion assays. Furthermore, siCREB transfection did not significantly affect the expression of vimentin compared with the siNC group in all 3 types of RCC cells (Fig. 5). These results suggested that there may be additional mechanisms by which CREB controls RCC function, however, the present study demonstrated that this does not occur via vimentin in vitro, as siCREB transfection did not significantly affect vimentin expression.

Using bioinformatics software, the authors previously demonstrated that that there is a putative CRE sequence in the promoter of fibronectin, and a putative binding site of CRE-binding protein is found in the promoter of vimentin (21). The present study aimed to determine whether there is a direct interaction between CREB and the CREsite in the fibronectin promoter or the CRE-binding protein site in the vimentin promoter. The current study employed siCREB, to target CREB mRNAs, and ChIP assays with the specific pCREB (Ser133) antibody were performed in 786-O (Fig. 6A), OS-RC-2 (Fig. 6B) and ACHN (Fig. 6C) cells. Compared with siNC treatment, siCREB treatment significantly decreased pCREB binding to the fibronectin promoter CRE sequence in all 3 cell lines (P<0.05; Fig. 6A-C) and did not significantly affect pCREB binding to the vimentin promoter CRE-binding protein site. The results of this experiment increased evidence for a direct interaction of pCREB (Ser133) with the fibronectin promoter in RCC, whilst confirming that pCREB (Ser133) does not target the vimentin promoter.