The human CRC cell line HT29 was maintained in McCoy's 5A medium and the CRC cell line SW1116 in RPMI-1640 medium (both from Gibco, Gaithersburg, MD, USA) supplemented with 10 % fetal bovine serum at 37°C in a 5% CO₂ incubator. For treatment with PD98059 (Cell Signaling Technology, Danvers, MA, USA), the cells were incubated with 20 µM PD98059 for 48 h before harvesting for assessments.

Total RNA was isolated using TRIzol reagent (Invitrogen/Gibco-BRL, Carlsbad, CA, USA). Total RNA (1 µg) was reverse transcribed using the PrimeScript RT reagent kit (Perfect Real-Time; Takara, Tokyo, Japan) to detect relative mRNAs. Relative quantitative data were obtained using the comparative Ct method on an Applied Biosystem 7900 quantitative PCR system (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. The specific primers for ZNF278 variants were as follows: variant 1 (GenBank accession no. NM_032050) F, 5′-GCAGTATCTGTAACCGAGAAGGC-3′ and R, 5′-ATTTCCCTTCAGGCCCCATG-3′; variant 2 (NM_032051) F, 5′-GAGGGTTGACAGTGGAAGGG-3′ and R, 5′-ATTTGGGGGCTCTGACATGG-3′; variant 3 (NM_032052) F, 5′-GCAGTATCTGTAACCGAGGTCTC-3′ and R, 5′-CGGACATGCACCTTCTGGAT-3′; variant 4 (NM_014323) F, 5′-AAAACCCACCACGGTGTTCC-3′ and R, 5′-CATTTCCCTTCAGGCCCCAT-3′. The Ct values obtained from different samples were compared using the 2^−ΔΔCt method. GAPDH served as an internal reference gene and the results were presented as the ratio of copies of target genes to GAPDH.

To construct the wild-type ZNF278 (GenBank accession no. NM_032050) expression vector, a PCR-generated full length ZNF278 cDNA was inserted into the EcoRI-HindIII sites of the expression vector CMV-MCS-3FLAG-SV40-Neomycin. Nested PCR was carried out to amplify the full-length ZNF278 cDNA. The following primers were used: F1, 5′-CGGCGCACCTGCGAGACTACAGA-3′ and R1, 5′-TCCCAGCAGTCCCCAGATGGTTGT-3′ for the first PCR; and F2, 5′-CCCAAGCTTCCATGGAGCGGGTGAAC-3′ and R2, 5′-CCGGAATTCTTTCCCTTCAGGCCCCAT-3′ for the second PCR. Before transfection, 5×10⁵ HT29 and SW1116 cells were seeded in 6 cm wells. The cells were transfected with 2 µg of either ZNF278 or control plasmid, using FuGENE HD transfection reagent (Promega, Madison, WI, USA), in accordance with the manufacturer's instructions. The cells were collected for measurements 48 h after transfection.

All transfections were performed using DharmaconFECT (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. ZNF278 small interfering RNA (siRNA) (sense, 5′-GCGCCGAUAUAAUGCUCUUTT-3′ and antisense, 5′-AAGAGCAUUAUAUCGGCGCGG-3′); and negative control siRNA (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) were designed and synthesized by GenePharma (Shanghai, China). ERK2 siRNA was sense, 5′-CACUUGUCAAGAAGCGUUAdTdT-3′ and antisense, 5′-CACUUGUCAAGAAGCGUUAdTdT-3′. Non-specific siRNAs (GenePharma) were used as the negative controls. The siRNA was complexed with the transfection reagent in a serum- and antibiotic-free medium for 6 h. After applying the transfection reagents, the cellular medium was replaced with the serum-containing maintenance medium, and the cells were incubated for 48 h. Gene expression silencing was confirmed by western blotting analysis.

Western blotting was performed according to the standard protocols, as previously described (8). The primary antibodies used in the present study were as follows: anti-ZNF278 (1:1,000; ab126903; Abcam, Cambridge, UK); anti-p-MEK1/2 (1:2,000; CST9121s); anti-p-ERK1/2 (CST4370s), anti-ERK1/2 (CST9102s), anti-MEK1/2 (CST9122s), anti-cyclin E1 (CST4129s), anti-cyclin D1 (CST2978s) and anti-p21 (CST2947T) (1:1,000) (all from Cell Signaling Technology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; KC-5G5; KangChen, Shanghai, China) was used as a loading control. The blots were analyzed using ImageJ 1.43 software and protein expression was normalized to GAPDH and relative to the control.

Cell viability was assessed by a tetrazolium salt (WST-8)-based colorimetric assay called Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Briefly, 5×10³ cells were transfected with ERK2 siRNA or negative control siRNA after 24 h of culture in 96-well plates. At the specified time-points, 10 µl of CCK-8 solution was added to each well and the cells were incubated for 1 h. Then, cell viability was determined by measuring the absorbance values at 450 nm using a microplate reader. Data are expressed as the percentage of viable cells, calculated as: relative viability (%) = [A450 (treated) - A450 (blank)]/[A450 (control) - A450 (blank)] × 100%.

Data are expressed as the mean ± SEM. Differences between two groups were compared using the Student's t-test. All experiments were repeated at least three times. P-values <0.05 were considered statistically significant.

To confirm which variants of ZNF278 were decreased after ZNF278-siRNA transfection, four pairs of specific primers were designed and synthesized. It was identified that all four ZNF278 variants were downregulated by ZNF278-siRNA transfection (Fig. 1A and B). As a previous study revealed overexpression of ZNF278 in the cells transfected with the pcDNA3.1-ZNF278 plasmid significantly increased the percentage of the S phase cells and decreased the percentage of the G0/G1 phase cells. Knockdown of ZNF278 expression significantly blocked the cell cycle at the G0/G1 phase (7). The cell cycle analysis revealed that G1 phase arrest was detected in SW1116 cells after depletion of ZNF278, suggesting that knockdown of ZNF278 induced cell cycle arrest. Furthermore, we examined the protein expression of cyclin E1 and D1 and p21, the three key genes involved in cell cycle progression (9,10). Expression of cyclin E1 and cyclin D1 were significantly decreased and p21 was increased after transfection of ZNF278 siRNA compared with the control siRNA in HT29 and SW1116 cells (Fig. 1C and D). Conversely, upregulation of cyclin E1 and D1 and downregultion of p21 were detected by ZNF278 plasmid transfection (Fig. 1E and F). The aforementioned data indicated that ZNF278 was involved in the regulation of CRC cell cycle progression.

We previously demonstrated that transfection of the SW1116 cells with pcDNA3.1-ZNF278 promoted cell growth, and ZNF278-siRNA transfection resulted in a significant inhibition of cell growth (7). In the present study, we further ensured this result in HT29 and SW1116 CRC cells using the CCK-8 assay. Knockdown of ZNF278 significantly inhibited cell proliferation compared to the control siRNA-transfected cells in both HT29 (Fig. 2A) and SW1116 (Fig. 2B) cells. Conversely, ectopic overexpression of ZNF278 accelerated cell proliferation in HT29 (Fig. 2C) and SW1116 cells (Fig. 2D). To elucidate the molecular mechanisms by which ZNF278-modulated CRC cell proliferation, we investigated the effects of knockdown of ZNF278 on the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, which is frequently aberrantly activated in human cancers and contributes to cell proliferation and survival (11,12). Western blotting revealed that transfection with ZNF278 siRNA significantly decreased ERK1/2 and MEK1/2 phosphorylation in both HT29 and SW1116 cells. However, no detectable changes in the total levels of ERK1/2 and MEK1/2 were observed (Fig. 3A and B). In contrast, ectopic overexpression of ZNF278 induced activation of ERK1/2 and promoted cell proliferation in HT29 and SW1116 cells (Fig. 4A and B). Furthermore, ZNF278 overexpression-induced CRC cell proliferation was markedly blocked by ERK2-siRNA transfection (Fig. 5). Thus, these data suggested that the ERK/MAPK pathway may participate in ZNF278-induced cell proliferation in CRC cells.

We demonstrated that downregulation of ZNF278 inhibited the ERK/MAPK pathway. To investigate whether reciprocal regulation of ZNF278 by the ERK-MAPK pathway occurs, we assessed the expression of ZNF278 in HT29 and SW1116 cells treated with PD98059 (a MEK1 inhibitor) or ERK2 siRNA using western blotting (13,14). Neither PD98059 nor ERK2 siRNA significantly affected the expression of ZNF278 in either HT29 or SW1116 cells (Fig. 6), which indicated that the ERK/MAPK signaling pathway may not participate in the regulation of ZNF278 expression.