In the present study, 49 RCC specimens and 49 peritumor specimens (control; ≥10 mm from the tumor edge) were obtained by surgical resection from patients who were pathologically confirmed for RCC, and admitted to the First Affiliated Hospital of Inner Mongolia Medical University (Hohhot, China) between 2011 and 2014. The ACHN human renal cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Gibco Eagle's minimum essential medium (EMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sijiqing, Hangzhou, China), 100 units/ml penicillin (CSPC Shijiazhuang Pharmaceutical Group, Shijiazhuang, China) and 100 mg/ml streptomycin (CSPC) in a humidified incubator under 5% CO₂ at 37°C.

To overexpress COP1 in the ACHN cells, the COP1 coding sequence was amplified with a human COPI cDNA clone (HG19467-CM; Sinobiological, Beijing, China) as template, and with COP1-specific primer pairs (forward, 5′-TCTAAGCTTATGTCTGGTAGCCGCCAGGCC-3′; reverse, 5′-ATAGGATCCTCATACCAATTCTAGCACCT-3′) under the following conditions: 95°C for 5 min, 35 cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 2 min followed by 72°C for 10 min. Subsequently, this was cloned into the eukaryotic pcDNA3.1(+) vector (Invitrogen; Thermo Fisher Scientific, Inc.). The COP1-pcDNA3.1(+) and control pcDNA3.1(+) plasmids were transfected into the ACHN cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The COP1-positive, ACHN(COP1+), and the control, ACHN(Con), cell clones were selected in the presence of 1 mg/ml G418 (Thermo Fisher Scientific, Inc.) and maintained in medium containing 0.7 mg/ml G418. To knockdown the COP1 expression in the ACHN(COP1+) cells, 25 or 50 nM COP1-specific small interfering (si)RNA, siRNA-COP1 or siRNA-Con (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was transfected with a HiPerFect Transfection Reagent (Qiagen, GmbH, Hilden, Germany) into the ACHN(COP1+) cells to abrogate COP1 expression.

RCC intra-tumor and peritumor tissue samples were homogenized and centrifuged at 3,000 × g at 4°C for 15 min to remove the non-homogenized tissue fraction. Each homogenate and ACHN cell sample was subjected to the lysis buffer in the Oligotex Direct mRNA Midi/Maxi kit (Qiagen, GmbH) for mRNA isolation and total mRNA isolation was performed according to the manufacturer's instructions. RT-qPCR was performed using the One-Step SYBR PrimeScript™ RT-PCR kit II (Perfect Real Time; Takara, Tokyo, Japan) to evaluate COP1, ETV1, matrix metalloproteinase 7 (MMP7) and β-actin mRNA expression levels. Each reaction system (25 µl for total volume) contained 12.5 µl SYBR Green PCR Master Mix, 2 µl template mRNA, 1 µl forward and reverse primer and 8.5 µl ddH₂O. The PCR reaction was performed under the following conditions: An initial denaturation for 5 min at 95°C, followed by 40 cycles of denaturation for 20 sec at 94°C, annealing for 20 sec at 61°C and extension for 20 sec at 72°C, then a final extension for 5 min at 72°C. The primer pairs (Sangon Biotech, Shanghai, China) were as follows: COP1, forward 5′-ATGAAATGACCTGCAATTCG-3′ and reverse 5′-TCACTGCTAGCTAACAGGTTC-3′; ETV1, forward 5′-CATCCCAGCAGAACAGAAGG-3′ and reverse 5′-CAGGTGTCATCATAAAACTGC-3′; MMP7, forward 5′-TCATAGAAATAATGCAGAAGC-3′ and reverse 5′-GTGAGTATTCTGCAACATCT-3′; β-actin, foward 5′-CATTAAGGAGAAGCTGTGCT-3′ and reverse 5′-GTTGAAGGTAGTTTCGTGGA-3′. Data were normalized to β-actin and fold changes were calculated using the 2^−ΔΔCq normalization method (17).

Western blot analysis was performed in RCC tissue specimens or ACHN cells to detect the protein levels of COP1, ETV1, MMP7 and β-actin. Briefly, each homogenized tissue sample or post-treated ACHN cell sample was lysed with ice-cold Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) and was centrifuged at 9,000 × g for 30 min at 4°C to remove the cellular debris. Proteins (25 µg) were separated by 12% SDS-PAGE gel (Sigma-Aldrich, St. Louis, MO, USA), and transferred to polyvinylidene fluoridehydrophobic membranes (EMD Millipore, Billerica, MA, USA), which were blocked with 2% bovine serum albumin (Ameresco, Inc., Framingham, MA, USA) at 4°C overnight. Subsequent to blocking, the membranes were incubated at 4°C for 2 h with rabbit polyclonal antibodies against COP1 (ab56400; 1:400; Abcam, Cambridge, UK), ETV1 (SAB2104467; 1:500; Sigma-Aldrich), MMP7 (AV46075; 1:500; Sigma-Aldrich) and β-actin (ab8227; Abcam) and washed three times. Following washes, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (7074; 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 4°C and washed three times. Finally, the specific binding band was detected with a chemiluminescent detection reagent (Amersham; GE Healthcare Life Sciences, Chalfont, UK). The relative expression levels for each protein were determined as a ratio to β-actin.

The growth curve of ACHN(COP1+) and ACHN(Con) cells was determined using the cell counting assay (18). Briefly, ACHN(COP1+) or ACHN(Con) cells were seeded in 12-well plates and were incubated at 37°C for 24, 48 or 72 h. The cells were trypsinized and counted using a hemocytometer (Reicher, Buffalo, NY, USA) and trypan blue (Sigma-Aldrich). A colony formation assay was conducted to evaluate the growth of ACHN(COP1+) and ACHN(Con) cells. ACHN(COP1+) and ACHN(Con) cells were seeded into a 12-well plate (200 cells/well) and 4 days later, the cell colonies were stained with 0.5% Crystal violet (Sigma-Aldrich) and counted directly by eye.

The migration and invasion capabilities of ACHN(COP1+) and ACHN(Con) cells were assessed using the scratch and transwell migration assays, respectively. For the scratch assay, ACHN(COP1+) and ACHN(Con) cells were grown in 6-well plates and, upon reaching 90% confluency, the plates were scratched using a 200-µl pipette tip. The healing process of the ACHN(COP1+) and ACHN(Con) cells was observed daily, and the migratory cells that crossed the wound area were counted at 48 h post inoculation. For the invasion assay, the experimental procedure followed was the same as the scratch assay. Briefly, cells were seeded (1×10⁵ cells) on the upper transwell chamber with the non-coated membrane (8-µm pore size; Merck Millipore, Darmstadt, Germany). Once the cells had grown to 2×10⁵ cells, they were coated with Matrigel (Sigma-Aldrich) and the lower chamber contained EMEM media with 20% FBS as a chemoattractant. The cells in the upper chamber were discarded after 24 h and the migratory cells in the lower chamber were counted using a microscope (BX60; Olympus Corporation, Tokyo, Japan). All the experiments were repeated in triplicate.

Statistical analysis was performed by unpaired t-test, using the Graphpad Prism software, version 5 (GraphPad Software, La Jolla, CA, USA) and P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of COP1 and its downstream factor, ETV1 in RCC, the expression levels of the two markers were determined in 49 RCC specimens. The RT-qPCR analysis indicated that the mRNA expression level of COP1 was significantly lower in the intratumor tissue samples (0.4704±0.0530) compared with the peritumor tissue samples (1.000±0.1208) of RCC (Fig. 1A; P<0.0001). The ETV1 mRNA levels were significantly increased in the intratumor tissue samples (4.931±0.637) when compared with the peritumor tissue samples (1.000±0.120) of RCC (Fig. 1B; P<0.0001). The down- and upregulated expression levels of COP1 and ETV1, respectively, were also confirmed at the protein level by western blot analysis (P<0.0001; Fig. 1C–E).

As demonstrated in Table I, no significant association was observed between gender or age and COP1 expression level in patients with RCC. However, COP1 expression was negatively associated with the tumor size (P=0.006), tumor-node-metastasis (TNM) stage (P=0.012), lymph node metastasis (P=0.010) and distant metastasis (P=0.023; Table I). The COP1 expression level in patients with larger tumors (>5 cm), lymph node metastasis, increased TNM stage or distant metastasis was significantly lower (Table I).

To further investigate the role of COP1 in the RCC cells, COP1 was overexpressed via a eukaryotic vector, pcDNA3.1(+). The coding sequence of COP1 was cloned into pcDNA3.1(+) and the recombinant plasmid was transfected into the AHCN cells. As demonstrated in Fig. 2A and B, the relative COP1 mRNA and protein levels were significantly increased in the AHCN cells transfected with the recombinant COP1-pcDAN3.1(+) plasmid compared with the AHCN(Con) cells (P<0.0001). The effect of COP1 on the growth of RCC AHCN cells was investigated by silencing COP1 expression. To knockdown COP1, AHCN(COP1+) cells were transfected with COP1-specific siRNA (siRNA-COP1). As demonstrated in Fig. 2C, the relative mRNA levels of COP1 were significantly reduced by transfection with 25 and 50 nM siRNA-COP1 compared with the siRNA-Con (P<0.01 and P<0.001, respectively). As demonstrated in Fig. 2D, the relative protein levels of COP1 were significantly reduced by the siRNA-COP1 transfection compared with the siRNA-Con in the AHCN(COP1+) and AHCN(Con) groups (P<0.0001 and P<0.01, respectively).

Following establishment of a successful knockdown model, the influence of COP1 overexpression on the growth of RCC AHCN cells was assessed using cell counting and colony formation assays. AHCN(COP1+) and AHCN(Con) cells were seeded with an initial titer of 10³ cells/ml in 12-well plates, and the cell number was counted every 24 h post-seeding. In a separate experiment, the seeded cells were transfected with siRNA-COP1 or siRNA-Con and the cell numbers were counted. As demonstrated in Fig. 2E, the titer of the AHCN(COP1+) cells was lower when compared with the AHCN(Con) cells; however, no significant difference was identified between the two groups. Furthermore, the transfection of siRNA-COP1 did not significantly influence the proliferation of AHCN(COP1+) cells (Fig. 2F; P>0.05). In addition, the overexpression of COP1 did not demonstrate a significant change in the colony numbers of AHCN cells (Fig. 3A and B). However, COP1 overexpression resulted in a significantly reduced colony size when compared with those of the AHCN(Con) group (Fig. 3C; P<0.05). These data indicate that COP1 overexpression exerts a limited influence on the growth of RCC cells.

It has been proposed that cell migration contributes to tumor metastasis (19). To further investigate the regulatory role of COP1 in the progression of RCC, the migration of AHCN(COP1+) and AHCN(Con) cells was assessed via scratch assay. The results demonstrated fewer migratory cells in the AHCN(COP1+) group when compared with the AHCN(Con) group (Fig. 4A and B; P<0.001). To further evaluate the invasion of AHCN(COP1+) and AHCN(Con) cells, the Matrigel-coated transwell assay was utilized. The results demonstrated a reduced number of invasive cells per field in the AHCN(COP1+) group when compared with the AHCN(Con) group (Fig. 4C; P<0.01). The results from the assays indicated that COP1 overexpression reduced the migration and invasion of RCC cells in vitro.

COP1 has been indicated to promote the degradation of ETV1 (14). To deduce the inhibition to the migration of AHCN cells by overexpressed COP1, the ETV1 and MMP7 expression levels in the AHCN(COP1+) and AHCN(Con) cells were assessed. No significant difference was observed in the ETV1 mRNA expression levels between the AHCN(COP1+) and AHCN(Con) groups (Fig. 5A; P<0.05). However, the MMP7 mRNA expression levels were significantly downregulated in the AHCN(COP1+) group compared with the AHCN(Con) group (Fig. 5B; P<0.01). Furthermore, the protein levels of the two markers were significantly downregulated in the AHCN(COP1+) group compared with the AHCN(Con) group (Fig. 5C–E; P<0.01). These data indicate that MMP7 is significantly inhibited by the overexpression of COP1.