TCGA (http://cancergenome.nih.gov) is a public database used to analyze the clinical significance of various genes. The UALCAN (https://ualcan.path.uab.edu/analysis.html) (27), Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/) (28) and Kaplan Meier plotter (https://kmplot.com/analysis/) (29) were used to assess the clinical relevance of genes in cancer based on the COAD dataset from TCGA database. The mRNA levels of ZNF695 and NEK2, and the Spearman's correlation coefficient between ZNF695 and NEK2 in patients with CRC were analyzed using the UALCAN and GEPIA databases. The association of ZNF695 with the survival of patients with CRC was analyzed using the Kaplan Meier plotter database.

The human normal colorectal cell line NCM460 was obtained from Hunan Fenghui Biotechnology Co., Ltd. (cat. no. CL0393). The CRC cell lines HCT-116 (cat. no. CCL-247) and HT-29 (cat. no. HTB-38) and were obtained from the American Type Culture Collection. The CRC cell line HCT-8 (cat. no. CL-0098) and 293T cells were purchased from Procell Life Science & Technology Co. Ltd. The cells were grown in RPMI 1640 complete medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution (Corning, Inc.). Cells were maintained in 6-well or 6-cm plates at 37°C in an incubator containing 5% CO₂.

The coding sequence of the human ZNF695 gene (NM_020394.5) was cloned into a pCDH vector (Addgene, Inc.). The lentivirus was produced using the 2nd generation system. Briefly, 293T cells were transfected with pCDH-Ctrl (10 µg, without the coding sequence of ZNF695) or pCDH-ZNF695 plasmids (10 µg), and the packaging plasmids PSPAX2 (7.5 µg; Addgene, Inc.) and PDM2G (2.5 µg; Addgene, Inc.) using VigoFect (cat. no. T001; Vigorous Biotechnology Beijing Co., Ltd.) at 37°C for 48 h. Subsequently, the culture supernatants were collected, filtered, ultracentrifuged (100,000 × g at 4°C for 2 h) and the lentivirus was harvested. HT-29 cells (8×10⁵) were infected with Ctrl and ZNF695 lentiviruses (20 µl) without detection of the titer, assisted by polybrene (8 µg/ml). After 48 h, puromycin (5 µg/ml) was used to select stable overexpressing cell lines for 1 week. After determining the overexpression efficacy by reverse transcription-quantitative PCR (RT-qPCR) and western blotting, the cells were immediately subjected to subsequent experiments.

Knockdown of ZNF695 and NEK2 was conducted using small interfering RNAs (siRNAs). Briefly, 8×10⁵ HCT-8 or HT-29 cells were seeded in 6-well plates and transfected with siRNAs (30 nM) against negative control, ZNF695 or NEK2 using RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h according to a previously described protocol (30). A total of 2 days after transfection the cells were subjected to other experiments, including RT-qPCR, western blotting and functional assays. siRNAs were obtained from Huzhou Hippo Biotechnology Co., Ltd. and the sequences were as follows: siCtrl, 5′-TTCTCCGAACGTGTCACGT-3′; siZNF695#1, 5′-GGATAGCTTCAATATGCAA-3′; siZNF695#2, 5′-CGATGTGAAGAATGTGGAA-3′; siNEK2, 5′-GGCTAGCTAGAATATTAAA-3′.

Total RNA was isolated from cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instruction. The RNA was subjected to RT using ReverTra Ace^™ qPCR RT Master Mix with gDNA Remover kit (Toyobo Co., Ltd.), according to the manufacturer's protocol. Firstly, 4X DN Master Mix with gDNA Remover was added to the RNA samples to remove gDNA at 37°C for 5 min. Subsequently, 5X RT M aster Mix II was added and RT was conducted as follows: 37°C, 15 min; 50°C, 5 min; 98°C, 5 min; and 4°C, hold. Subsequently, qPCR was performed using SYBR master mixture (cat. no. QPK-201; Toyobo Co., Ltd.) on a Bio-Rad system (Bio-Rad Laboratories, Inc.). The qPCR procedure was performed as follows: One cycle at 37°C for 30 sec; followed by 40 cycles at 94°C for 5 sec and 60°C for 30 sec; and a final dissociation stage of one cycle at 94°C for 15 sec, 60°C for 30 sec and 94°C for 15 sec. The primer sequences used for qPCR were: ZNF695, forward 5′-ATCTCCCTTGGTGAGGATAGC-3′, reverse 5′-GACAAAACTGAGTGTTTGGCTG-3′; NEK2, forward 5′-TGCTTCGTGAACTGAAACATCC-3′, reverse 5′-CCAGAGTCAACTGAGTCATCACT-3′; β-actin, forward 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse 5′-CTCCTTAATGTCACGCACGAT-3′. The relative expression levels of the indicated gene were calculated using the 2-^∆∆Cq method (31).

Total protein was isolated from the cells using RIPA buffer (Beyotime Institute of Biotechnology) and the concentration of total proteins was detected using a BCA kit (Thermo Fisher Scientific, Inc.). Equal amounts (30–50 µg) of total proteins in loading buffer were then separated by SDS-PAGE on 10 or 12% gels and transferred onto PVDF membranes. Subsequently, the membranes were incubated with 5% skimmed milk for 1 h at room temperature, washed with PBS-0.1% Tween 20 (PBST) three times, and incubated with primary antibodies at 2–8°C overnight. After further washing with PBST three times, the membranes were incubated with secondary antibodies for 2 h at room temperature. Protein abundance was detected using the ECL-Plus kit (Amersham; Cytiva). The following primary antibodies were used: ZNF695 (cat. no. 25556-1-AP; 1:800; Proteintech Group, Inc.), NEK2 (cat. no. 24171-1-AP; 1:800; Proteintech Group Inc.), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)-Akt (cat. no. 4060; 1:1,000; Cell Signaling Technology, Inc.), ribosomal protein S6 (S6; cat. no. 2217; 1:1,000; Cell Signaling Technology, Inc.), p-S6 (cat. no. 2211; 1:1,000; Cell Signaling Technology, Inc.), p21 (cat. no. 2947; 1:1,000; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. 3498; 1:1,000; Cell Signaling Technology, Inc.), β-actin (cat. no. 66009-1-Ig; 1:5,000; Proteintech Group Inc.) and GAPDH (cat. no. 60004-1-Ig; 1:5,000; Proteintech Group Inc.). The mouse anti-rabbit IgG-HRP secondary antibody (cat. no. sc-2357; 1:10,000) was purchased from Santa Cruz Biotechnology, Inc. and the HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (cat. no. SA00001-1; 1:10,000) were was from Proteintech Group, Inc.

Cell viability and proliferation was assessed by detecting the relative viability of CRC cells using the Cell Counting Kit-8 (CCK8; cat. no. C0039; Beyotime Institute of Biotechnology) kit. Briefly, 3,000-4,000 HCT-8 or HT-29 cells were seeded in triplicate into 96-well plates, which contained 200 µl complete culture medium. The detection and calculation methods were carried out as previously described (30). To detect the cytotoxicity of rapamycin (cat. no. S1039; Selleck Chemicals), 3,000-4,000 HCT-8 or HT-29 cells were seeded in triplicate into 96-well plates, supplied with 200 µl complete culture medium. After 6–8 h, the cells adhered on the plates and cell culture medium was removed. Subsequently, 200 µl complete culture medium containing different concentrations of rapamycin (0, 0.1, 0.5, 2 and 5 nM) was added into each well. After incubating the cells at 37°C for 48 h, cell viability was detected using the CCK8 kit. The growth inhibition rate of 0.1/0.5/2/5 nM rapamycin was calculated as follows: Growth inhibition rate (%)=(OD450 value of 0 nM rapamycin-OD450 value of 0.1/0.5/2/5 nM rapamycin)/OD450 value of 0 nM rapamycin ×100.

After ZNF695 knockdown, a total of 1,500 siCtrl, siZNF695#1 and siZNF695#2 HCT-8 cells were seeded into 6-well plates, and were cultured in complete culture medium for 7 days. After ZNF695 overexpression with or without NEK2 knockdown, a total of 1,000 Ctrl, ZNF695-overexpressing HT-29 cells and ZNF695 + siNEK2 cells were seeded into 6-well plates, and were cultured in complete culture medium for 9 days. Subsequently, the culture medium was removed and the plates were washed three times with PBS. The colonies were then fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Images of the colonies were collected using a camera (Canon EOS R100; Canon, Inc.).

A total of 1×10⁶ HCT-8 cells transfected with siRNAs (siCtrl, siZNF695#1 and siZNF695#2) and HT-29 cells transfected with overexpression vectors (Ctrl and ZNF695) were seeded into 6-well plates. For apoptosis analysis, the cells were trypsinized in EDTA-free trypsin, and were stained with PI and Annexin V-FITC (cat. no. 40302ES60; Shanghai Yeasen Biotechnology Co., Ltd.) for 10 min at room temperature For cell cycle analysis, the cells were trypsinized and harvested in 70% iced alcohol. After 16–24 h, the cells were stained with PI and RNaseA (cat. no. 40301ES60; Shanghai Yeasen Biotechnology Co., Ltd.) for 30 min at room temperature. Apoptosis and cell cycle analyses were performed on a flow cytometry system (model no. A00-1-1102; Beckman Coulter, Inc.) at an excitation wavelength of 488 nm, and were analyzed by CytExpert software (Beckman Coulter, Inc.).

The coding sequence of ZNF695 and Flag were synthesized and cloned into pCDNA3.1 vectors (Addgene, Inc.). Subsequently, 1×10⁷ HT-29 cells in a 10-cm plate were transfected with 15 µg ZNF695-Flag-pCDNA3.1 plasmids using VigoFect at 37°C for 72 h. After 2 days, the cells were collected and subjected to ChIP-qPCR experiments using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (cat. no. 9005; Cell Signaling Technology, Inc.), according to the manufacturer's instructions. The cells were divided into the ChIP-IgG group and the ChIP-Flag group; the IgG group served as the internal control. ChIP-IgG (cat. no. 2729; Cell Signaling Technology, Inc.) or ChIP-Flag (cat. no. 14793; Cell Signaling Technology, Inc.) was used during immunoprecipitation. qPCR was performed as aforementioned to quantify the amount of DNA in each group, and the qPCR primer sequences for the promoter of the NEK2 gene were: Forward, 5′-CCACTTGCAGTGCCTTACATAA-3′ and reverse, 5′-GGAATTGCCAACCAGGAGAA-3′. The qPCR procedure was performed as follows: One cycle at 37°C for 30 sec; followed by 40 cycles at 94°C for 5 sec and 60°C for 30 sec; and a final dissociation stage of one cycle at 94°C for 15 sec, 60°C for 30 sec and 94°C for 15 sec.

Caspase-Glo reagent (Promega Corporation) was used to detect the activity of caspase 3/7, according to the manufacturers' instructions. The method was carried out as previously described (30).

The promoter sequence (−2,000-0 bp) of the NEK2 gene was cloned into the pGL3 basic vector (Promega Corporation). The Renilla luciferase vector, pCMV-RL-TK (Promega Corporation) was applied to determine the transfection efficiency. For ZNF695 knockdown, HCT-8 cells were co-transfected with siRNAs (siCtrl, siZNF695#1 and siZNF695#2) and the luciferase vectors. For ZNF695 overexpression, HT-29 cells were co-transfected with overexpression vectors (Ctrl and ZNF695) and the luciferase vectors (pGL3 vectors with NEK2 promoter and pCMV-RL-TK vectors) using Lipofectamine^® 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, luciferase activity was detected using an the Dual-Luciferase^® Reporter Assay System (cat. no. E1910; Promega Corporation). The relative luciferase activity was normalized to Renilla luciferase.

GraphPad Prism (version 8.0; Dotmatics) was used to analyze the data (mean ± standard error) and to determine the statistical differences. Adobe Illustrator 2021 (Adobe Systems, Inc.) was used to generate the figures. Unpaired Student's t-test was applied to determine the difference between two groups. One-way ANOVA, followed by Tukey's post hoc test, was applied to compare the differences among groups. Kaplan-Meier survival curve and the log-rank test were applied to analyze overall survival. P<0.05 was considered to indicate a statistically significant difference.

First, the clinical significance of ZNF695 in CRC was analyzed by assessing the transcript abundance of ZNF695 in data from TCGA database. It was shown that ZNF695 mRNA expression was upregulated in the tumor tissues of most cancer types compared with non-cancer tissues (adjacent tissues from patients with cancer), including CRC (Fig. 1A and B). In addition, the cancer tissues from patients with stage 1–4 CRC exhibited higher ZNF695 expression than normal tissues (Fig. 1C). The methylation levels on the promoter of the ZNF695 gene were not significantly reduced in CRC tissues from patients of all stages compared with those in normal tissues (Fig. 1D). Notably, the cancer tissues from patients with stage 2 CRC only exhibited significantly decreased methylation levels on the promoter of the ZNF695 gene compared with those in normal samples (Fig. 1E). Furthermore, the prognostic value of ZNF695 for patients with CRC was analyzed using the Kaplan Meier plotter database. Using the Kaplan-Meier plotter, when setting the ‘best cutoff, OS, 18 months’, the patients with high expression of ZNF695 (n=406) tended to have a shorter survival time than those with low ZNF695 expression (n=655) (Fig. 1F), but the difference was not statistically significant (P=0.065). Subsequently, experiments were conducted to assess the expression of ZNF695 in NCM460 normal colorectal cells and in CRC cells. Compared with those in NCM460 cells, the mRNA levels of ZNF695 were increased in HT-29, HCT-8 and HCT116 CRC cells (Fig. 1G). Taken together, these results indicated that ZNF695 may participate in CRC development.

Next, whether overexpression of ZNF695 contributes to the malignant proliferation of CRC cells was investigated; to address this, HCT-8 and HT-29 cells were used. HCT-8 is a stem-like cell line, which has characteristics of fast proliferation and poor differentiation (32,33), whereas HT-29 is a moderately differentiated colonic cancer cell line (33). Using these two cell lines could demonstrate the response of CRC cells to the effect of ZNF695 expression on proliferation. ZNF695 was knocked down in HCT-8 cells by transfecting the cells with siZNF695. Notably, the mRNA and protein levels of ZNF695 were significantly reduced in siZNF695#1 and siZNF695#2 HCT-8 cells compared with those in siCtrl cells (Fig. 2A). Furthermore, knockdown of ZNF695 suppressed the proliferation of HCT-8 cells, as demonstrated by the CCK8 assay (Fig. 2B). To validate the oncogenic function of ZNF695, ZNF695 was overexpressed in HT-29 cells, and the overexpression efficacy was confirmed by RT-qPCR and western blotting (Fig. 2C). Notably, overexpression of ZNF695 promoted the proliferation of HT-29 cells (Fig. 2D). Furthermore, it was shown that ZNF695 knockdown inhibited the colony formation of HCT-8 cells, whereas the opposite results were observed in HT-29 cells with overexpression of ZNF695 (Fig. 2E and F). Thus, the in vitro results suggested that ZNF695 may be critical for the of CRC cells.

To assess whether ZNF695 regulates the cell cycle progression of CRC cells, the cells were subjected to knockdown or overexpression of ZNF695 and then underwent PI staining, followed by cell cycle analysis with flow cytometry. It was found that ZNF695 knockdown resulted in a higher percentage of HCT-8 cells in G₀/G₁ phase and a lower percentage in G₂/M phase compared with those in the siCtrl group (Fig. 3A and C). By contrast, overexpression of ZNF695 exhibited the opposite effect on the cell cycle progression of HT-29 cells (Fig. 3B and C). Next, the cells were stained with Annexin V/PI and apoptosis was analyzed by flow cytometry. The results showed that knockdown of ZNF695 induced the apoptosis of HCT-8 cells compared with in the siCtrl group (Fig. 3D and F). Consistently, overexpression of ZNF695 suppressed the apoptosis of HT-29 cells (Fig. 3E and F). In addition, the activity of caspase 3/7 was inhibited by ZNF695 overexpression and increased by ZNF695 knockdown (Fig. 3G). Subsequently, the expression levels of the apoptosis markers p21 and Bcl-2 were assessed in cells with knockdown or overexpression of ZNF695 by western blotting. The results supported the indication that ZNF695 can suppress the apoptosis of CRC cells (Fig. 4). Collectively, these findings suggested that ZNF695 may be involved in regulating the cell cycle and apoptosis of CRC cells.

To investigate the downstream effector of ZNF695 in CRC, the present study firstly analyzed the genes positively correlated with ZNF695 in patients with CRC. Spearman correlation results showed that C17orf53, UBE2T, NEK2, CKS1B and BIRC5 were the most significantly correlated genes with ZNF695 in patients with CRC (Fig. 5A). Previous studies have demonstrated that upregulation of NEK2 contributes to the development of various types of cancer, including CRC (34–36); therefore, the current study explored whether ZNF695 contributed to CRC progression by regulating NEK2. To address this RT-qPCR and western blotting were performed. It was shown that the abundance of NEK2 mRNA was downregulated in CRC cells with ZNF695 knockdown, whereas it was upregulated when ZNF695 was overexpressed (Fig. 5B and C). Dual luciferase reporter assay results revealed that the luciferase activity of the NEK2 gene promoter was reduced in CRC cells with ZNF695 knockdown, whereas it was increased in CRC cells overexpressing ZNF695 (Fig. 5D and E). The ChIP assay also showed that ZNF695 activated the expression of the NEK2 gene (Fig. 5F). Based on TCGA database, there was a positive correlation between ZNF695 and NEK2 in CRC samples (Fig. 5G). Furthermore, the transcript levels of NEK2 were enhanced in CRC tissues compared with those in non-cancer tissues (Fig. 5H). These findings indicated that ZNF695 may positively regulate the expression of NEK2 in CRC cells and in patients with CRC.

NEK2 is a kinase and thus has the potential to enhance the phosphorylation of certain proteins. The Akt/mTOR signaling pathway is hyper-activated in various types of cancer, including CRC (37). Therefore, whether ZNF695 regulates the activity of the Akt/mTOR signaling pathway in CRC cells was investigated. Western blotting showed that ZNF695 knockdown reduced the phosphorylation levels, but not the protein levels, of Akt and S6 in HCT-8 cells (Fig. 6A), whereas the opposite results were observed in HT-29 cells with overexpression of ZNF695 (Fig. 6D). Subsequently, the cells were treated with different concentrations of the mTOR inhibitor, rapamycin. It was shown that different doses of rapamycin exhibited reduced cytotoxic effects and increased growth inhibition in cells with ZNF695 knockdown as compared with siCtrl HCT-8 cells (Fig. 6B and C). In addition, HT-29 cells with ZNF695 overexpression exhibited higher sensitivity and growth inhibition comparing with Ctrl HT-29 cells in response to treatment with rapamycin at different doses (Fig. 6E and F). These results indicated that ZNF695 may activate the Akt/mTOR signaling pathway and the expression of ZNF695 dictates the sensitivity of CRC cells to rapamycin treatment.

Finally, whether upregulation of NEK2 supports the proliferation of CRC cells driven by ZNF695 was investigated by knocking down NEK2 in CRC cells. The western blotting results showed that NEK2 was efficiently silenced by siRNAs in HT-29 cells (Fig. S1). NEK2 was knocked down in CRC cells with overexpression of ZNF695. Western blotting results showed that NEK2 was efficiently silenced by siRNA (Fig. 7A). CCK8 and colony formation assay results revealed that the accelerated proliferation of HT-29 cells was inhibited by knockdown of NEK2 (Fig. 7B and C). Furthermore, the activity of caspase 3/7 was enhanced by silencing NEK2 in ZNF695-overexpressing cells (Fig. 7D). These results indicated that ZNF695 may contribute to CRC cell proliferation via the upregulation of NEK2.