Expression data for ZNF667-AS1 across 33 types of cancer and corresponding normal tissues were extracted from TCGA (http://sangerbox.com) and genotype-tissue expression databases (http://sangerbox.com) and subsequent pan-cancer analyses of ZNF667-AS1 concerning overall survival (OS), disease-specific survival (DSS), immune infiltration (TIMER, EPIC, IPS, MCPcounter, xCELL, QUANTISEQ and CIBERSORT), genomic heterogeneity and stemness (DNA, EREG-METH, DMPs, ENHs, RNAss and EREG-EXPs) were conducted via the SangerBox web tool (http://sangerbox.com) (16).

The OC cell line SKOV3 used in the present study was purchased from Procell Life Science & Technology Co., Ltd. The IOSE80 and OVCA433 cells were a gift from the First Affiliated Hospital of the Anhui Medical University (Anhui, China). SKOV3, IOSE80 and OVCA433 cells were cultured in McCoy's 5A medium (Procell Life Science & Technology Co., Ltd), RPMI-1640 medium, or RPMI-1640 medium (Procell Life Science & Technology Co., Ltd), respectively, supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained at 37˚C in a humidified 5% CO₂ incubator and the medium was renewed every 2 days.

In vitro transfection of OC cells was performed using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) with a PCDH vector expressing lncRNA ZNF667-AS1 (2.5 µg of lncRNA ZNF667-AS1 and 5 µl of Lipofectamine^® 2000 reagent), ZNF667-AS1 small interfering (si)RNA (10 µl of siRNA and 5 µl of Lipofectamine^® 2000 reagent), or corresponding negative controls (Wuhan Miaoling Biotech Science Co., Ltd.) and the transfection mass of the control group was the same as that of the experimental group. The sequences of plasmid were shown in Table SI. Transfections were conducted at 60% confluence in 6-well plates following the manufacturer's protocol. Following transfection, the experiment was continued for 24-48 h in a 37˚C cell incubator.

Total RNA was extracted from 2x10⁶ cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and detection of RNA concentration and purity (260/280) was performed using nucleic acid protein analyzer. RNA was reverse transcribed into cDNA with a Promega reverse transcription system (Promega Corporation). RT-qPCR was performed with SYBR Green Master Mix (Vazyme Biotech Co., Ltd.) on a real-time PCR detection system: 95˚C predenaturation 30 sec, 40 cycles of 95˚C 10 sec and 60˚C 30 sec. Relative lncRNA ZNF667-AS1 levels were quantified by the 2^-ΔΔCq method and normalized to the level of GAPDH (17). The RT-qPCR primers used are listed in Table SI. In studies that do not involve metabolic regulation, GAPDH, as a widely expressed steward gene, is an internal reference gene with stable expression and is often used in lncRNA studies (18). RNA extraction, cDNA synthesis, and qPCR performed according to the manufacturer's protocols and these experiments were replicated three times.

Cell proliferation was assessed using the EdU incorporation assay. Briefly, cells grown on coverslips in 24-well plates were incubated with EdU medium for 2 h at 37˚C. Following fixation in 4% paraformaldehyde at room temperature for 15 min and permeabilization in 0.3% Triton X-100 (Biosharp Life Sciences), EdU labelling was performed via a click reaction cocktail for 30 min, following the manufacturer's instructions (Beyotime Institute of Biotechnology). The cells were counterstained with Hoechst 33342 (Beyotime Institute of Biotechnology) at room temperature for 10 min and visualized under a fluorescence microscope (magnification, x100).

The cells were seeded in 6-well plates and cultured for 10-14 days at 37˚C with 5% CO₂. Colonies were fixed in 4% paraformaldehyde at room temperature for 15 min, stained with 0.1% crystal violet solution (Beyotime Institute of Biotechnology) at room temperature for 15 min and counted manually. A total of three wells in each group were counted and used as separate experiments.

Confluent cell monolayers in 6-well plates were scratched with a 200 µl pipette tip to create wound gaps, washed three times with PBS and incubated in serum-free medium. Wound closure was monitored by taking phase contrast images at 0 and 24 h post wounding using an inverted microscope (magnification, x100).

Cell migratory and invasive capacities were evaluated via Transwell assays. For invasion, the upper chamber was precoated with Matrigel matrix at 37˚C for 2 h (BD Biosciences). Cells in serum-free medium were added to the upper chamber, while complete medium supplemented with 10% FBS as a chemoattractant was added to the lower well. Following 24-48 h of incubation, the cells on the lower surface were fixed in 4% paraformaldehyde at room temperature for 15 min and stained with crystal violet solution (Beyotime Institute of Biotechnology). The cells were imaged and quantified under a light microscope (magnification, x100). For the migration assays, the upper chamber was without the Matrigel matrix.

RNA extracted from three pairs of stably transfected ZNF667-AS1 overexpressing and control OC cells was subjected to RNA-seq analysis by Genergy Bio-Technology (Shanghai) Co. Differentially expressed genes were identified and analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. The gene set for the KEGG pathway analysis was obtained from the Molecular Signatures Database (curated gene sets, canonical pathways, KEGG analysis for human gene symbols; v2022.1; https://www.gsea-msigdb.org/gsea/index.jsp).

FISH was performed using a Shanghai GenePharma Co., Ltd. kit, according to the manufacturer's protocol. OC cells grown on coverslips were hybridized with a Cy3-labelled ZNF667-AS1 probe, counterstained with DAPI and visualized via confocal microscopy (magnification, x400). The ZNF667-AS1 probe was designed and synthesized by Shanghai GenePharma Co., Ltd.

Total protein was extracted from the cells with RIPA lysate (Beyotime Institute of Biotechnology) and determined by BCA. The proteins (20-100 µg) were subjected to SDS-PAGE (10%) and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk for 1 h at room temperature and then incubated with primary and secondary antibodies. Bound antibodies were detected via enhanced chemiluminescence (ECL). The specific antibodies employed in this experiment were GAPDH (cat. no. 200306-7E4; OriGene Technologies, Inc.), Tubulin (cat. no. 250009; OriGene Technologies, Inc.), PCNA (cat. no. 10205-2-AP; Proteintech Group, Inc.), MMP2 (cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (cat. no. 10375-2-AP; Proteintech Group, Inc.), rabbit second antibody (cat. no. SA00001-2; Proteintech Group, Inc.) and mouse second antibody (cat. no. SA00001-1; Proteintech Group, Inc.). All primary antibodies were diluted 1:1,000 and incubated at 4˚C for 12 h, while goat anti-rabbit and anti-mouse IgG H&L (HRP) second antibodies were diluted 1:5,000 and incubated at room temperature for 1 h. Finally, ImageJ (National Institutes of Health) was used to calculate the gray value of the protein strip.

The data are presented as the means ± standard deviation. Statistical comparisons between groups were conducted via unpaired Student's t-test in GraphPad Prism 8.0 (Dotmatics) and R version 4.1.2 (http://www.R-project.org/). P<0.05 was considered to indicate a statistically significant difference.

The present study explored the prognostic significance of lncRNA ZNF667-AS1 in different types of cancer by using data from the SangerBox website. The analysis revealed that high expression of lncRNA ZNF667-AS1 was associated with poor prognosis in several types of cancer, particularly glioblastoma (GBM) and skin cutaneous melanoma. For example, in GBM patients, elevated lncRNA ZNF667-AS1 is associated with reduced OS, as shown in Fig. 1A, where a forest plot demonstrates a significant difference (P<0.05) between the high- and low-expression groups. By contrast, in uveal melanoma and OC, high expression of lncRNA ZNF667-AS1 is linked to an improved prognosis and the forest plot in Fig. 1B demonstrated improved outcomes in patients with elevated expression.

The present study subsequently analyzed the correlation between lncRNA ZNF667-AS1 and various immune cell types by using the SangerBox website. To investigate the infiltration levels of immune cells, the present study employed several algorithms, including TIMER, EPIC, IPS, MCPcounter, xCELL, QUANTISEQ and CIBERSORT. These algorithms assess the composition and activity of immune cells in the tumor microenvironment from different perspectives. For example, TIMER focuses on estimating tumor-infiltrating lymphocytes, whereas MCPcounter distinguishes the abundance of different immune cell subpopulations. As shown in Fig. 2, multiple algorithms indicated that higher expression of lncRNA ZNF667-AS1 was associated with increased immune cell infiltration in tumors such as colon adenocarcinoma and thyroid carcinoma, whereas lower expression was associated with decreased immune cell infiltration in tumors such as brain lower grade glioma. These findings indicated a potential role for this lncRNA in regulating the tumor immune microenvironment. The present study further investigated the effect of genomic heterogeneity on the expression of lncRNA ZNF667-AS1. The findings indicated that lncRNA ZNF667-AS1 had a complex relationship with various metrics of genomic heterogeneity, including mutations, copy number variations and chromosomal structural variations in different types of cancer. Fig. 3 illustrated the intricate correlations between lncRNA ZNF667-AS1 expression and these heterogeneous parameters and highlights its potential role as a marker of tumor evolution and treatment response.

Tumor stemness, a key factor in cancer biology, refers to the ability of tumor cells or tissues to maintain or acquire stem cell characteristics. This information is crucial for understanding tumor biology, predicting disease prognosis and developing new anticancer treatments. As shown in Fig. 4, in most types of cancer, lncRNA ZNF667-AS1 expression was negatively associated with tumor stemness and this relationship was confirmed by several methods, including DNA, EREG-METH, DMPs, ENHs, RNAss and EREG-EXPs. These results showed that lncRNA ZNF667-AS1 may prevent tumor cells from acquiring stem-like properties.

Analysis of TCGA data using the SangerBox platform revealed downregulation of lncRNA ZNF667-AS1 across multiple types of cancer, including OC (Fig. 5A). The present study validated this finding in ovarian cell lines; lower ZNF667-AS1 levels occurred in the SKOV3 and OVCA433 cancer lines than in normal IOSE80 cells. ZNF667-AS1 expression was particularly diminished in OVCA433 cells (Fig. 5B).

To elucidate the functional role of ZNF667-AS1 in OC, the present study generated ZNF667-AS1-overexpressing OVCA433 cells and ZNF667-AS1-knockdown SKOV3 cells via plasmid transfection and siRNA strategies, respectively. Successful overexpression and knockdown were confirmed by RT-qPCR (Fig. 5C and D). A colony formation assay revealed significantly reduced proliferation in ZNF667-AS1-overexpressing OVCA433 cells compared with controls. Conversely, ZNF667-AS1 knockdown enhanced SKOV3 cell proliferation (Fig. 6A-D). The EdU experiment showed consistent results (Fig. 6E-H). In addition, western blotting revealed that overexpression of ZNF667-AS inhibited the expression of the proliferation marker PCNA (Fig. 7E).

The present study further examined the effects of ZNF667-AS1 on OC cell motility via Transwell and wound healing assays. Wounds were introduced into layers of SKOV3-SI, OVCA433-OE and corresponding control cells by scratching and the cells were subsequently cultured for 24 h. Overexpression of the lncRNA ZNF667-AS1 significantly decreased the spreading potential of OVCA433 cells, whereas downregulation of the lncRNA ZNF667-AS1 significantly increased the spreading potential of SKOV3 cells (Fig. 7A and B). The Transwell assay revealed a similar trend with respect to the invasion and migration abilities of SKOV3 and OVCA433 cells (Fig. 7C and D). In addition, overexpression of ZNF667-AS inhibited the expression of the migration and invasion markers MMP2 and MMP9 (Fig. 7E).

The FISH results (Fig. 8A and B) revealed that lncRNA ZNF667-AS1 was mainly expressed in the nuclei of SKOV3 and OVCA433 cells. To elucidate the molecular mechanisms by which ZNF667-AS1 functions in OC, RNA-seq was performed on ZNF667-AS1-overexpressing and control OVCA433 cells. Differentially expressed genes were identified using volcano plot and heatmap analyses (Fig. 8C and D). KEGG pathway enrichment analysis revealed significant enrichment of differentially expressed genes in the TNF pathway (Fig. 8E and F). TNF-α is expressed mainly on the cell membrane, but its signaling process involves gene expression regulation in the nucleus (19) and ZNF667-AS1 is expressed mainly in the nucleus. Western blotting confirmed that overexpression of ZNF667-AS1 decreased the expression of TNF (Fig. 7E).

Missense mutations were the predominant type among the differentially expressed genes, with NOTCH4 being the most frequently mutated gene in OC (Fig. 8G). These results suggested that ZNF667-AS1 was a key regulator of the TNF signaling cascade in OC.