ZNF263 promotes gastric cancer cell growth by regulating TXNDC9 transcription

2.1 Bioinformatics analysis

Survival data for GC patients were obtained from the Kaplan Meier Plotter database, provided the survival curve, ZNF263 expression levels in GC tissues were analyzed using the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) database.

2.2 Cell culture and transfection

Human gastric cancer cell lines MNK-45 and AGS were obtained from the Chinese Academy of Sciences Type Culture Collection. Cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 95% humidified environment at 37 °C and 5% CO₂.

For gene knockdown and overexpression studies, si-ZNF263 (GTGCAACATTTGCGGAAAA), negative control siRNA (si-NC), ZNF263, and TXNDC9 overexpression plasmids were transfected using Lipofectamine 2000.

2.3 Cell viability

Cells were planted in 96-well plates with 1 × 10³ cells per well. After transfection, add 10 μL of CCK8 reagent to each well and incubate for 2 hours at 37 °C. Every 24 hours, the optical density (OD) was measured at 450 nm with an enzyme microplate reader.

2.4 Cell proliferation

Cells in logarithmic growth phase were seeded into 96-well plates and treated with EdU medium. The medium was discarded, and cells were rinsed with Phosphate Buffered Saline (PBS). The cells were then treated with 4% paraformaldehyde before being removed. Glycine was added, and the cells were shaken for 5 minutes to decolorize them. The glycine solution was removed, and the cells were washed again with PBS. Permeabilization was performed using TritonX-100 in PBS, and cells were incubated for 10 minutes. The Apollo staining reaction solution was added, and the cells were incubated in the dark at room temperature for 30 minutes. Hoechst 33342 was used to stain the nuclei, and the cells were incubated in the dark at room temperature for 10 minutes. The cells were studied, photographed, and quantified using a fluorescence microscope.

2.5 Apoptosis

GC cells were planted in 6-well plates and incubated for 48 hours. Cells were collected with trypsin without Ethylenediaminetetraacetic acid (EDTA), washed with PBS, and stained with Annexin V-Fluorescein isothiocyanate (FITC) and Propidium Iodide (PI) in the dark for 15 minutes at room temperature using the Annexin V-FITC/PI Apoptosis Detection Kit. Flow cytometry was used to assess cellular apoptosis.

2.6 Western blotting

The proteins were isolated from GC cells using Radioimmunoprecipitation Assay (RIPA) lysis buffer. The protein concentration was measured using the Bicinchoninic Acid Assay (BCA) Protein Assay Kit to establish the electrophoresis loading volume. Protein samples were combined with loading buffer before being denatured in a water bath at 95 °C. Proteins were separated using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), then transferred to a Polyvinylidene Fluoride (PVDF) membrane and blocked with 5% milk in Tris Buffered Saline with Tween-20 (TBST). After washing with TBST, primary antibodies were applied to the membrane, which was then incubated overnight at 4 °C. Secondary antibodies were added after washing with TBST and incubated for 2 hours at room temperature. Membranes were imaged with a gel imaging device, and densitometry was done with ImageJ software. Antibodies used: cleaved-caspase 3 (Abcam, Cambridge, UK, ab2302, 1:500); cleaved-PARP (Abcam, Cambridge, UK, ab32064, 1:1000); ZNF263 (Abcam, Cambridge, UK, ab129100, 1:1000).

2.7 Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from GC cells using TRIzol reagent, and its purity and concentration were determined using a Nanodrop 1000 spectrophotometer. cDNA was produced using a reverse transcription kit. The qRT-PCR was carried out using an Applied Biosystems 7300 Real-Time PCR System (Foster City, CA, USA). TXNDC9 gene expression was standardized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression levels were obtained using the 2^−ΔΔCt technique. TXNDC9 forward primer: 5′-CTGCTTCAGACTACCAAACTGG-3′, TXNDC9 reverse primer 5′-CTCTGTAGAAATGGCAAACCACA-3′.

2.8 Chromatin immunoprecipitation (ChIP)

The Simple ChIP Enzymatic Chromatin IP kit was used as directed by the manufacturer to accomplish chromatin immunoprecipitation. HEK293T cells were cross-linked with 1% formaldehyde for 10 minutes, then quenched with glycine for 5 minutes at room temperature before being lysed. Lysates were treated with anti-ZNF263 antibody or normal rabbit Immunoglobulin G (IgG). DNA was purified after reverse DNA cross-linking using Sodium Chloride (NaCl) and protease. DNA samples were analyzed by qRT-PCR to assess TXNDC9 promoter binding.

2.9 Dual-luciferase reporter assay

TXNDC9 and ZNF263 binding sites (wild-type or mutant) were cloned into the pGL3 reporter vector. Using Lipofectamine® 3000, these reporter plasmids were co-transfected with ZNF263 and NC into HEK293T cells in 24-well plates. Luciferase activity was determined using the Dual Luciferase Reporter Gene Assay Kit.

2.10 Statistical analysis

Data from three independent experiments are presented as mean ± Standard Deviation (SD). The statistical analysis was carried out using SPSS 22.0 (IBM Corp, Armonk, NY, USA). An unpaired Student’s t-test was used to compare the two groups. For comparisons across groups, one-way Analysis of Variance (ANOVA) was employed, followed by Tukey’s post hoc test. Statistical significance was considered as p < 0.05.

3.1 ZNF263 is highly expressed in GC

To assess ZNF263 expression in GC tissue and its impact on patient survival, we analyzed ZNF263 expression using the UALCAN database and investigated its association with patient survival. The results revealed that GC patients had significantly higher ZNF263 expression compared to normal tissues (Fig. 1A), and this overexpression was correlated with poorer survival outcomes (Fig. 1B). These findings suggest that elevated ZNF263 expression in GC is associated with a worse prognosis.

Fig. 1.ZNF263 is highly expressed in GC. (A) UALCAN database query ZNF263 expression in GC patient tissues. (B) The influence of ZNF263 expression on GC patient survival rate as determined by a Kaplan Meier Plotter database query. ***p < 0.001. ZNF: Zinc finger protein; TCGA: The Cancer Genome Atlas; STAD: Stomach Adenocarcinoma; HR: Hazard Ratio.

3.2 ZNF263 promotes GC cell growth

GC cells were transfected with either si-ZNF263 or a ZNF263 overexpression plasmid. Western blot analysis confirmed successful transfection, with increased ZNF263 expression in the overexpression group and reduced expression in the si-ZNF263 group si-ZNF263 group (Fig. 2A). The cell viability was evaluated using the CCK8 test (Fig. 2B), and cell proliferation was evaluated by EdU experiment (Fig. 2C,D). The results demonstrated that ZNF263 significantly increased the proportion of EdU-positive cells and enhanced cell viability, while si-ZNF263 dramatically decreased both parameters. Therefore, ZNF263 promotes GC cell growth.

Fig. 2.ZNF263 promotes GC cell growth. (A) Western blot analysis of ZNF263 protein expression in transfected cells. (B) CCK8 assay for cell viability. (C) EdU detection of cell proliferation. (D) Quantification of EdU-positive cells. *p < 0.05, **p < 0.01, ***p < 0.001. ZNF: Zinc finger protein; si-NC: negative control siRNA; EdU: 5-Ethynyl-2′-deoxyuridine; DAPI: 4′,6-Diamidino-2-phenylindole.

3.3 ZNF263 inhibits apoptosis in GC cells

Flow cytometry was used to measure GC cell apoptosis rate. Our findings demonstrated that si-ZNF263 significantly increased both the cleaved-caspase3 and cleaved-PARP protein expressions, as well as the apoptosis rate. In contrast, ZNF263 group exhibited a significant reduction in both cell apoptosis rate (Fig. 3A) and the protein levels of cleaved-caspase3 and cleaved-PARP (Fig. 3B). These findings suggest that ZNF263 inhibits apoptosis in GC cells.

Fig. 3.ZNF263 inhibits apoptosis in GC cells. (A) Flow cytometry detection of cell apoptosis rate. (B) Western blot analysis of cleaved-caspase3 and cleaved-PARP expressions in cells. **p < 0.01, ***p < 0.001. ZNF: Zinc finger protein; si-NC: negative control siRNA; PI: Propidium Iodide; FITC: Fluorescein isothiocyanate; PARP: Poly(ADP-ribose) polymerase.

3.4 ZNF263 promotes TXNDC9 transcription

The hTFtarget database predicted that TXNDC9 is a downstream target of ZNF263. Sequence analysis of the TXNDC9 promoter region with ZNF263 using the JASPAR database (Fig. 4A) indicated that ZNF263 might function as a transcription factor for TXNDC9. The top three binding sites were selected for further study (Fig. 4B). ChIP-qPCR confirmed that ZNF263 was recruited to the E1 site of the TXNDC9 promoter region, but not the E2 and E3 sites (Fig. 4C). Dual luciferase reporter assays demonstrated that ZNF263 greatly increased the luciferase activity in the Wild Type (WT)-TXNDC9 area but had no effect on the Mutant (MUT)-TXNDC9 (Fig. 4D). qPCR analysis showed that si-ZNF263 significantly reduced the TXNDC9 mRNA expression in GC cells, whereas ZNF263 increased the TXNDC9 mRNA expression (Fig. 4E). These results confirm that ZNF263 acts as a transcription factor for TXNDC9. Furthermore, overexpression of TXNDC9 reversed the inhibitory effects of si-ZNF263 on GC cell survival and increased apoptosis (Fig. 4F–H).

Fig. 4.ZNF263 promotes TXNDC9 transcription. (A) ZNF263 binding motif found in the JASPAR database. (B) Three sites binding to the TXNDC9 promoter. (C) ChIP-qPCR confirmed the recruitment of ZNF263 to the TXNDC9 promoter sites. (D) Dual-luciferase reporter gene assay confirmed the interaction between ZNF263 and the TXNDC9 promoter. (E) PCR analysis of TXNDC9 mRNA expression following transfection with si-ZNF263 or ZNF263 in GC cells. (F) Western blot analysis of TXNDC9 protein expression in transfected cells. (G) CCK8 assay for cell viability. (H) Flow cytometry detection of cell apoptosis rate. **p < 0.01, ***p < 0.001. TXNDC9: Thioredoxin domain-containing protein 9; ZNF: Zinc finger protein; TSS: Transcription Start Site; IgG: Immunoglobulin G; si-NC: negative control siRNA; PI: Propidium Iodide; FITC: Fluorescein isothiocyanate; WT: Wild Type; MUT: Mutant.