The IHC staining of a human CRC tissue microarray was conducted by Servicebio Biotechnology Co., Ltd. The paired CRC tissues and adjacent normal tissues (at a distance of 5 cm from the tumor margin) were collected from patients with CRC undergoing surgical treatment between September, 2022 and March, 2023. The inclusion criteria were patients with primary pathologically diagnosed CRC. The exclusion criteria were patients with CRC had been treated with hormonal therapy or chemoradiotherapy, or patients with chronic inflammatory disease, severe cerebro-cardiovascular disease, respiratory disease, liver or renal disease, or female patients who were pregnant. The tissue sections (5-µm-thick) were subjected to IHC staining for ZNF169 and ANKZF1. Briefly, the sections were deparaffinized in xylene and hydrated in a graded alcohol series. Antigen retrieval and endogenous peroxidase blocking were performed using citrate buffer (pH 6) (cat no. G1219, Wuhan Servicebio Technology Co., Ltd.) and 3% hydrogen peroxide (cat no. 88597, MilliporeSigma), respectively. The slides were incubated with 10% goat serum (BIOSS) for 1 h and with rabbit polyclonal ZNF169 antibody (1:1,000 dilution; cat no. B-10117, Weibo Biotechnology Co., Ltd.) and rabbit polyclonal ANKZF1 primary antibody (1:500 dilution; cat no. 20446-1-AP, Proteintech Group, Inc.) at 4°C overnight. The antigen-antibody complex was detected with a biotinylated goat anti-rabbit antibody (1:2,500 dilution; cat no. BA1003, Boster Biological Technology Co., Ltd.) conjugated with streptavidin-horseradish peroxidase was used to detect the antigen-antibody complex. Visualization was conducted using nickel-enhanced 3,3-diaminobenzidine tetrahydrochloride (Beijing Solarbio Science & Technology Co., Ltd.). The tissue sections were then counterstained with hematoxylin (cat no. G1004, Wuhan Servicebio Technology Co., Ltd.) for 3–5 min at room temperature. For the mean density analysis of IHC staining, three or more fields (magnification, ×200) were randomly selected from each section for imaging. The staining density value is the relative expression level of the proteins. The high or low expression of ZNF169 and ANKZF1 was set during the density analysis. The present study was approved by the Ethics Committee of Beijing Rehabilitation Hospital of Capital Medical University (Beijing, China). A form of written informed consent was obtained from each participant in the present study.

The transcript level of ZNF169 and ANKZF1, the Spearman's correlation analysis of the correlation between ZNF169 and other genes, as well as the prognostic significance of ZNF169 and ANKZF1 in patients with CRC were analyzed based on TCGA database (http://cancergenome.nih.gov).

The 293T cells (cat no. CRL-3216) was purchased from the American Type Culture Collection. The normal human colorectal cell line NCM460 (cat no. CL0393) was purchased from FENGHUIBIO (www.fenghbio.cn). The CRC cell lines, RKO (cat no. CL-0196), HCT-116 (cat no. CL-0096), HT-29 (cat no. CL-0118), SW620 (cat no. CL-0225) and HCT-8 (cat no. CL-0098), were purchased from Procell Life Science & Technology Co. Ltd. STR profiling was used to confirm the authentication of the HT-29 cell line. All the cells were cultured in RPMI-1640 complete medium (Gibco; Thermo Fisher Scientific, Inc.), containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution (Corning, Inc.). The cells were maintained in 6-cm plates at 37°C in an incubator with 5% CO₂.

For the knockdown assay, the HCT-116, RKO or HT-29 CRC cells were transfected with small interfering RNAs (siRNAs) against ZNF169, ANKZF1 or negative control siRNA using RNAiMAX regent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 3 µl of 20 µM siRNAs and 4 µl RNAiMAX were added to 400 µl Opti-MEM (cat no. 31985070, Gibco; Thermo Fisher Scientific, Inc.) and were incubated at room temperature for 15 min. The mixture was then added to 1.6 ml culture medium. After 48 h, the cells were examined using reverse transcription-quantitative PCR (RT-qPCR), western blot analysis, cellular function assays and other experiments. The siRNAs were purchased from HIPPOBIO company (http://www.hippobiotec.com/). The siRNA sequences were as follows: siCtrl, 5′-UUCUCCGAACGUGUCACGU-3′; siZNF169, 5′-GAAGCUCCAAGAUGCUCUAGU-3′; siANKZF1, 5′-GGUGCUAUAUUUCAAGGAAGA-3′.

For the overexpression assay, the coding sequences of ZNF169 or ANKZF1 were synthesized and cloned into the pCDH overexpression plasmids by HIPPOBIO company (http://www.hippobiotec.com/). Subsequently, lentiviruses were produced by transfecting the 293T cells using VigoFect (cat no. Vigorous Biotechnology, http://www.vigorousbiol.com/index.htm) with pCDH-Ctrl, pCDH-ZNF169, or pCDH-ANKZF1 vectors, alongside the packaging plasmids, including PSPAX2 and PDM2G. After 48 h, the virus supernatants were harvested, filtered and concentrated. Subsequently, the lentiviruses were added to the medium, supplied with polybrene, to infect the HT-29 or HCT-116 cells for 48 h. Finally, the cell lines with a stable overexpression were constructed by treating the cells with puromycin (cat no. HB-PU-1000, HANBIO, http://www.hanbio.net/) for 2 weeks.

Total RNA was extracted from the HCT-116, RKO or HT-29 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. ReverTra Ace™ qPCR RT Master Mix with gDNA Remover kit (cat no. FSQ-301, Toyobo Life Science) was then applied to reverse transcribe the RNA into cDNA, according to the manufacturer's instructions. Subsequently, the quantification of cDNA was performed using qPCR with SYBR master mixture (Takara Bio, Inc.) on a Bio-Rad system (Bio-Rad Laboratories, Inc.). The PCR thermocycling conditions were as follows: i) Holding stage, 95°C for 3 min; ii) cycling stage, 95°C for 15 sec; iii) Cycling stage, 60°C for 30 sec; iv) cycling stage, 72°C for 30 sec; cycling stage, 2–4; 40 cycles. The qPCR primer sequences were as follows: ANKZF1 forward, 5′-CGGTTTAACCTAAAGCAACGTCT-3′ and reverse, 5′-CCGATCCAGTGTCTGCAAGT-3′; β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′. The quantification of qPCR data was performed according the 2^−ΔΔCq method (28).

Total protein was extracted from the NCM460, RKO, HCT-116, SW620, HT-29 and HCT-8 cells using RIPA buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using the BCA kit (Thermo Fisher Scientific, Inc.). A total of 40–70 µg proteins were then separated by SDS-PAGE on 10 or 12% gels, followed by transfer onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibodies at 4°C overnight. After washing with PBS (Wuhan Servicebio Technology Co., Ltd.)-Tween (Beyotime Institute of Biotechnology), the membranes were incubated with secondary antibodies for 2 h at room temperature. The protein signal was detected using the ECL-Plus kit (cat no. RPN2232, Cytiva). The antibody against ZNF169 (1:1,000; cat. no. ab225924) was purchased from Abcam, and the antibodies against ANKZF1 (1:800; cat. no. 20447-1-AP) and GAPDH (1:10,000; 10494-1-AP) were obtained from Proteintech Group, Inc. The mouse anti-rabbit IgG-HRP secondary antibody (1:8,000; cat. no. sc-2357) was from Santa Cruz Biotechnology, Inc.

The proliferation of the CRC cells was determined using a CCK-8 kit. Briefly, a total of 3,000 HCT-116, RKO and HT-29 cells were seeded into 96-well plates, which contained 200 µl culture medium. After 8 h, 20 µl CCK-8 regent (cat no. C0039, Beyotime Institute of Biotechnology) were added to each well and incubated at 37°C for 3 h. The OD value was then measured at 450 nm, which represented the relative cell viability of 0 h. At the time points of 24, 48, 72 and 96 h, the cell viability was sequentially determined to compare to the OD450 value at 0 h.

Following ZNF169 knockdown, 2,000 HCT-116 and RKO cells from the siCtrl and siZNF169 groups were seeded into six-well plates, and the cells in completed culture medium were maintained for 8 days in a cell incubator at 37°C. Following the overexpression of ZNF169, 500 HT-29 cells from the Ctrl and ZNF169 overexpression groups were cultured in six-well plates for 10 days. After colonies were formed, they were washed twice with PBS (Wuhan Servicebio Technology Co., Ltd.). Subsequently, methanol was used to fix the colonies for 30 min at room temperature and 0.1% crystal violet (Beyotime Institute of Biotechnology) was used to stain them for 30 min at room temperature. Finally, the images of the colonies were captured using a camera (EOS700D, Canon, Inc.).

Caspase-3/caspase-7 activity was detected using the Caspase-Glo reagent (Promega Corporation). Briefly, 10,000 suspended HCT-116, RKO or HT-29 cells were seeded into 96-well plates, to which 100 µl Caspase-Glo was added. After 90 min, the caspase-3/caspase-7 activity was detected using a microplate reader (M2009PR, Tecan infinite, Tecan Group, Ltd.).

To assess DNA synthesis, the HCT-116, RKO or HT-29 CRC cells were subjected to EdU staining using the BeyoClick™ EdU Cell Proliferation kit with Alexa Fluor 555 (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. A total of 5×10⁵ HCT-116 and RKO cells transfected with a negative control siRNA or a siRNA against ZNF169, or a total of 4×10⁵ HT-29 cells infected with an empty control or ZNF169 overexpression lentivirus, were seeded onto coverslips in six-well plates. After 24 h, the cells were incubated with EdU reagent at 37°C for 4–6 h. The cells on the coverslips were then washed with PBS, fixed with 4% paraformaldehyde (Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature, treated with 0.5% Triton X-100 (Beyotime Institute of Biotechnology) for 15 min at room temperature, and finally stained with Click Additive Solution (Beyotime Institute of Biotechnology) for 30 min at room temperature and DAPI (Beyotime Institute of Biotechnology) for 15 min at room temperature in the dark. Images of the EdU-positive cells were captured under a microscope and quantified by Photoshop.

The promoter of ANKZF1 was cloned into the pGL3 basic vector (Promega Corporation). The HCT-116 and HT-29 cells were seeded in 24-well plates and co-transfected with the aforementioned luciferase vectors and the expression vectors (knockdown or overexpression vector) using VigoFect for 48 h in a cell incubator at 37°C. The Renilla luciferase vector, pCMV-RL-TK (Promega Corporation), was used as an internal control to determine the transfection efficiency. At 48 h following transfection, the dual luciferase activity was checked by using the Dual-Luciferase^® Reporter Assay System (E1910, Promega Corporation), according to the manufacturer's protocols.

A ChIP assay was performed to determine whether ZNF169 binds to the promoter of the ANKZF1 gene in the control and ZNF169-overexpressing cells. The ChIP-qPCR assay was performed using the SimpleChIP enzymatic chromatin IP kit (cat no. 26156, Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The purified DNA was then subjected to qPCR analysis. The qPCR primers were designed to amplify the promoter sequence (2,000 bp upstream of the ANKZF1 transcript) of the ANKZF1 gene. The qPCR primer sequences were as follows: Forward, 5′-CTCCATGTTCTTCCATCAACT-3′ and reverse 5′-CTCCTTATTGGCAGGTGGTC-3′.

GraphPad Prism 8.0 software (Dotmatics) was used for statistical analyses. The results are presented as the mean ± standard error of the mean (meean ± SEM). Adobe Illustrator 2021 (Adobe Inc.) was utilized to create the figures. The Chi-squared test (χ² value) was performed to assess the relevance of ZNF169 expression in CRC and normal samples. An unpaired Student's t-test and one-way ANOVA followed by Tukey's post hoc test were applied to compare the differences between two groups and among more than two groups, respectively. P<0.05 was considered to indicate a statistically significant difference.

To examine the clinical relevance of ZNF169 in CRC, the present study first collected data from TCGA and analyzed the expression of ZNF169 in CRC and normal tissues. It was found that ZNF169 expression was upregulated in CRC tissues compared with normal tissues (Fig. 1A). GEPIA is a newly developed interactive web server which uses the log-rank test (the Mantel-Cox test) for survival analysis (29). The patients with CRC were subsequently divided into the ZNF169 high and low expression groups, and their survival rates were analyzed from GEPIA. The patients in the high ZNF169 expression group had a shorter survival time (Fig. 1B), suggesting that ZNF169 functions as a negative prognostic biomarker for patients with CRC. In order to validate the protein expression of ZNF169, a tissue microarray containing cancer and normal tissues from patients with CRC was subjected to the IHC staining of ZNF169. The results indicated that the protein expression of ZNF169 was higher in CRC tissues than in normal tissues (Fig. 1C and Table I). It was also found that ZNF169 was highly expressed in CRC cell lines, including RKO, HT-29 and HCT-116, as compared with the NCM460 normal cell line (Fig. 1D).

The present study then explored the cellular function of ZNF169 in CRC cells. As the HCT-116 and RKO cells had a relatively higher expression of ZNF169, these cells were transfected with a negative control siRNAs and with siRNAs against and ZNF169. The results of western blot analysis revealed that ZNF169 was efficiently silenced in the siZNF169 cells (Fig. 2A). The results of the CCK-8 assay demonstrated that ZNF169 knockdown suppressed the proliferation of the HCT-116 and RKO cells (Fig. 2B). Consistently, the colony formation capacity of the HCT-116 and RKO cells was also suppressed following the knockdown of ZNF169 (Fig. 2C). Subsequently, the HT-29 cells were transfected with an empty control or ZNF169 overexpression lentivirus, and the results of western blot analysis validated the overexpression efficacy (Fig. 2D). The results of CCK-8 and colony formation assays demonstrated that the ectopic overexpression of ZNF169 significantly promoted the malignant growth of the HT-29 cells (Fig. 2E and F). These results suggested that ZNF169 may be critical for the growth and proliferation of CRC cells.

To examine the mechanisms through which ZNF169 contributes to the proliferation of CRC cells, the CRC cells in which ZNF169 was knocked down or overexpressed were subjected to EdU staining, which predicts DNA synthesis. It was found that ZNF169 knockdown significantly suppressed the proportion of EdU-positive cells in both cell lines (Fig. 3A and B). By contrast, the ectopic overexpression of ZNF169 enhanced the proportion of EdU-positive HT-29 cells (Fig. 3A and B). The present study then analyzed the activity of caspase-3/7, which can be used to predict apoptosis, following the knockdown or overexpression of ZNF169 in the cells. As shown in Fig. 3C, ZNF169 knockdown enhanced caspase-3/7 activity in the HCT-116 and RKO cells. Conversely, ZNF169 overexpression suppressed caspase-3/7 activity in the HT-29 cells. These results indicated that ZNF169 promotes DNA synthesis and suppresses the apoptosis of CRC cells.

Since ZNF169 serves as a transcription factor in eukaryotes, there must be downstream effectors that participate in the ZNF169-induced promotion of CRC cell proliferation. Therefore, the present study then analyzed the genes positively correlated with ZNF169 in CRC tissues based on TCGA database. A total of 10 of the most significantly positively correlated genes, including ZNF789 and ANKZF1, are illustrated in Fig. 4A. Since the overexpression of ANKZF1 has been reported to be associated with the poor prognosis of patients with CRC (25,26), the present study aimed to explore whether ZNF169 regulated ANKZF1 in the present study, and we aim to explore the significance of other genes positively correlated with ZNF169, such as ZNF789 and AGAP4, in the subsequent studies.. Based on the results of RT-qPCR and western blot analysis, it was found that ZNF169 knockdown markedly suppressed the mRNA and protein expression levels of ANKZF1 in the HCT-116 and RKO cells (Fig. 4B and C). By contrast, the overexpression of ZNF169 potentiated the expression of ANKZF1 in the HT-29 cells (Fig. 4B and C). In order to examine whether ZNF169 regulates the transcriptional activity of the ANKZF1 gene, the promoter sequence of the ANKZF1 gene was cloned into a pGL3.basic vector and a dual luciferase activity assay was performed. It was demonstrated that ZNF169 knockdown inhibited the luciferase activity of the ANKZF1 promoter, whereas ZNF169 overexpression promoted it (Fig. 4D). In addition, a ChIP-qPCR assay was carried out to examine the interaction between the ZNF169 protein and the promoter sequence of the ANKZF1 gene. It was found that the promoter sequence of the ANKZF1 gene was highly enriched in the ANKZF1-Flag group of CRC cells (Fig. 4E). Collectively, ZNF169 may potentiate the transcriptional activity of the ANKZF1 gene by directly interacting with its promoter sequence.

As ZNF169 was found to promote CRC cell proliferation and positively regulate the expression of ANKZF1, the present study then aimed to determine whether ANKZF1 participates in CRC growth. To this aim, overexpression and knockdown assays of ANKZF1 were performed in the CRC cells. The results of RT-qPCR revealed that ANKZF1 could be efficiently silenced by siANKZF1 when compared with siCtrl in the HT-29 cells (Fig. S1A). Thus, ANKZF1 was knocked down (Fig. S1B) in ZNF169-overexpressing CRC cells and the cells were subjected to CCK-8 and colony formation assays. The results of western blot analysis revealed that ANKZF1 was efficiently silenced after the ZNF169-overexpressing cells were transfected with siANKZF1 (Fig. 5A). The results of CCK-8 and colony formation assays demonstrated that ANKZF1 knockdown suppressed the growth and proliferation of the CRC cells in which ZNF169 was overexpressed (Fig. 5B and C). To validate the function of ANKZF1, ANKZF1 was then overexpressed in the HCT-116 cells in which ZNF169 was silenced. ANKZF1 overexpression lentivirus significantly increased the mRNA level of ANKZF1 in the HCT-116 cells when compared with the cells transfected with the Ctrl lentivirus (Fig. S1B). Based on the results of western blot analysis, ANKZF1 was found to be overexpressed in the HCT-116 cells in which ZNF169 was silenced (Fig. 5D). ANKZF1 overexpression restored the proliferation and growth of the HCT-116 cells in which ZNF169 was knocked down (Fig. 5E and F). These results suggested that ZNF169 may contribute to the growth and proliferation of CRC cells through the upregulation of ANKZF1.

Finally, the clinical relevance of ANKZF1 was investigated in patients with CRC. Based on TCGA database, ANKZF1 expression was upregulated in the cancer tissues of patients with CRC compared with normal tissues (Fig. 6A). Consistently, the results of IHC staining revealed that the protein expression of ANKZF1 was higher in CRC tissues compared with adjacent normal tissues (Fig. 6B). It was also demonstrated that there was a positive correlation between ZNF169 and ANKZF1 expression in the tissues of patients with CRC (Fig. 6C and Table II). Importantly, a high expression of ANKZF1 was associated with a poor prognosis of patients with CRC as compared with those with a low expression of ANKZF1 (Fig. 6D). Thus, ANKZF1 may be considered a negative prognostic biomarker for patients with CRC.