Epigenetic-Mediated Downregulation of Zinc Finger Protein 671 (ZNF671) Predicts Poor Prognosis in Multiple Solid Tumors

Zinc finger protein 671 (ZNF671) is a member of the largest transcription factor family in the human genome. However, the methylation status, expression, and prognostic role of ZNF671 in solid tumors remain unclear. The aim of this study was to explore the relationship between ZNF671 and the prognosis of patients with solid tumors. We performed a pan-cancer analysis of the methylation status and mRNA and protein expression of ZNF671 using The Cancer Genome Atlas (TCGA) database and the Human Protein Atlas. We further evaluated the prognostic value of ZNF671 expression among numerous cancer types using the “Kaplan–Meier plotter” (KM plotter) database. We found that downregulation of ZNF671 is associated with hypermethylation of its promoter. Survival analysis established that the downregulation of ZNF671 predicts poor prognosis in breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), and uterine corpus endometrial carcinoma (UCEC) solid tumors. CCK-8 and Transwell functional assays showed that ZNF671 could inhibit tumor cell proliferation, migration, and invasion. These results indicate that ZNF671 is an excellent predictive factor for BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC solid tumors and may play crucial roles in the development and progression of these tumors.


INTRODUCTION
Epigenetic regulation is essential for the normal development and maintenance of gene expression in mammals. Many biological processes are involved in DNA methylation, histone modification, and nucleosome remodeling (1). Disruption of epigenetic processes can lead to activation of oncogenes and/or the inactivation of tumor suppressor pathways, and the accumulation of epigenetic changes in the molecular landscape is a hallmark of cancer (2). DNA methylation is an early event in tumorigenesis and plays a major role in tumor initiation and progression (3). Therefore, it is important to identify reliable prognostic and predictive biomarkers in solid tumors that may help in early diagnosis and treatment strategies.
In this study, we systematically analyzed the methylation level and expression of ZNF671 and explored its correlation with solid tumors by analyzing The Cancer Genome Atlas (TCGA) database and the Human Protein Atlas (HPA). We evaluated the prognostic role of ZNF671 in 18 solid tumors using the Kaplan-Meier (KM) plotter database. Our results provide important insights into ZNF671 hypermethylation as a promising biomarker for eight solid tumors and thereby provide novel perspectives for the treatment of tumors.

TCGA Dataset Analysis
Gene methylation and expression data were downloaded from TCGA (http://cancergenome.nih.gov) and were interpreted, normalized, and log2 scaled using the online analysis tool. This study was approved by the Institutional Ethical Review Boards of Guangzhou Medical University Cancer Center.

Immunohistochemistry Analysis
The HPA database (https://www.proteinatlas.org/) was used to perform a pan-cancer analysis of ZNF671 protein expression (14). ZNF671 was entered in the database (https:// www.proteinatlas.org/) to acquire ZNF671 expression for individual tumors of each cancer type (Scar bar = 200µm). All IHC images have been manually annotated by certified pathologists.

Prognostic Analysis
An online KM plotter database was used to assess the correlation of intra-individual ZNF671 mRNA expression with progression-free survival (15). ZNF671 was entered in the database (http://kmplot.com/analysis/) to acquire pan-cancer KM survival plots. Hazard ratio (HR), 95% confidence intervals, and log-rank P were determined and presented on the main plots.

CCK-8 and Transwell Assays
For the CCK-8 assay, cells (1 × 10 3 ) were seeded into 96-well plates, incubated for 0-4 days, stained with CCK-8 (Dojindo, Tokyo, Japan), and absorbance values were determined at 450 nm using a spectrophotometer. Transwell migration and invasion assays were carried out using Transwell chambers (8 µm; Corning, Tewksbury, MA, USA) without or with Matrigel (BD Biosciences, San Jose, CA, USA). Cells were added to the upper chamber in serum-free medium, and the lower chamber contained culture medium with 20% FBS. The cells were incubated for 12 or 24 h at 37 • C in 5% CO 2 and were then fixed and stained. Cells on the undersides of the filters were observed and counted under i200 magnification.

Statistical Analysis
Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Differences between two groups were analyzed using the two-tailed unpaired Student's t-test; P < 0.05 was considered statistically significant.

ZNF671 Promoter Is Hypermethylated in Several Solid Tumor Types
To determine the methylation level of ZNF671 across cancer types, we analyzed human pan-cancer methylation data in the TCGA database. Among the 18 cancers examined, the promoter of ZNF671 was hypermethylated in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell

ZNF671 Protein Expression Is Downregulated in Several Solid Tumor Types
We performed a pan-cancer analysis of the protein expression of ZNF671 using the HPA, which presented the protein expression of ZNF671 in 17 different tumor types. As shown in Figure 4A, the protein expression level of ZNF671 was downregulated in most cancers. Statistical analysis indicated that ZNF671 expression was low and/or absent in urothelial (75%), breast (90.9%), cervical (90%), endometrial (83.3%), head and neck (100%), renal (100%), liver (77.8%), lung (100%), pancreatic (72.7%), prostate (100%), skin (100%), and thyroid (75%) cancers. ZNF671 protein expression was low and/or absent in only 41.7 and 45.4% of colorectal and stomach cancers, respectively (Figure 4B), and the protein expression of ZNF671 in sarcoma was unclear. These results indicate that ZNF671 protein expression is downregulated in urothelial, breast, cervical, endometrial, heck and neck, renal, liver, lung, pancreatic, prostate, skin, and thyroid cancers and that ZNF671 may play a tumor suppressor role in cancer progression.

ZNF671 Is a Prognostic Biomarker in Several Solid Tumor Types
To further assess the prognostic role of ZNF671 in different cancers, we explored the relationship between mRNA expression of ZNF671 and patient survival using an online tool (http:// kmplot.com). High The relationship between ZNF671 methylation, mRNA and protein expression, and prognosis among different cancer types is summarized in Table 1. Overall, the results indicate that epigenetic-mediated downregulation of ZNF671 predicts poor survival in BRCA, CESC, HNSC, KIRP, LUAD, PAAD, UCEC, and SARC and that ZNF671 may play a tumor suppressor role in tumor progression, which is consistent with previous studies in HNSC, UCEC, and KIRP (11,16,17).

ZNF671 Inhibits Tumor Cell Proliferation, Migration, and Invasion
To assess the effects of ZNF671 in BRCA, CESC, LUAD, PAAD, and SARC, we performed CCK-8 and Transwell assays using MCF7, Hela, PC9, PANC1, and HOS cells transfected with ZNF671 or vector. As shown in Figure 6A, Western blot analysis validated that ZNF671 protein level was obviously elevated after transfection of ZNF671 plasmid in MCF7, Hela, PC9, PANC1, and HOS cells. Overexpression of ZNF671 suppressed the proliferation ( Figure 6B) and migratory and invasive (Figures 6C-E) abilities of the tumor cells as determined by Transwell migration and invasion assays. These findings indicate that ZNF671 inhibits the proliferation and migratory and invasive abilities of BRCA, CESC, LUAD, PAAD, and SARC cells in vitro.

DISCUSSION
ZNF671 belongs to the KRAB-ZF family of transcription factors, which contain C2H2-type ZFs and a Krüppel-associated box (KRAB) domain. KRAB-ZFPs are involved in regulating cell differentiation, proliferation, apoptosis, tumor suppression, and immune control (7,9,11,18,19). Our previous studies demonstrated that ZNF671 is a tumor suppressor that is epigenetically silenced by DNA methylation in nasopharyngeal carcinoma, but there is limited information regarding the role of ZNF671 methylation among different solid cancer types. The present study found that downregulation of ZNF671 was associated with its promoter hypermethylation among many different cancer types. Functional assays and prognostic analysis found that high ZNF671 expression suppressed tumor cell proliferation, migration, and invasion, and downregulation of ZNF671 was associated with poor clinical prognosis in BRCA, CESC, HNSC, KIRP, LUAD, PAAD, and UCEC. Thus, ZNF671 represents a novel predictive marker for the prognosis of patients with BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC.
In this study, we found that the ZNF671 promoter is significantly methylated in 17 solid tumors, based on the TCGA datasets, which is consistent with studies in urothelial carcinoma, clear cell renal cell carcinomas, and cervical cancer (16,17,20). Next, we compared and analyzed the mRNA and protein expression levels of ZNF671 in these solid tumors. Statistical analysis revealed that the expression of ZNF671 was negatively correlated with its promoter hypermethylation in BLCA, BRCA, CESC, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, READ, SARC, SKCM, and UCEC, suggesting a crucial role for ZNF671 in the progression of these cancer types.
To further explore the potential prognostic value of ZNF671 in BLCA, BRCA, CESC, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, READ, SARC, SKCM, and UCEC, we used KM plotter analysis to evaluate the correlation between ZNF671 expression and survival probability. KM plotter is used to analyze individual genes in different cancers (21). We found that low ZNF671 expression may predict poorer survival in BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC. CCK-8 and Transwell migration and invasion assays showed that ZNF671 could inhibit tumor cell proliferation, migration, and invasion. However, additional studies are still needed to explore the molecular mechanism underlying the effects of ZNF671 In conclusion, this is the first report to systematically evaluate the correlation between methylation of ZNF671 and prognosis among 18 solid tumors. The prognostic signature observed in our study was linked to hypermethylation of ZNF671. Our findings reveal that ZNF671 is hypermethylated in BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC, and functional assays established that ZNF671 inhibits tumor cell proliferation, migration, and invasion. Further prognosis analysis indicated that high expression of ZNF671 was associated with longer overall survival (OS) and that ZNF671 expression is a favorable prognostic indicator in BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC, but the mechanism remains unknown. Further studies are needed to clarify this issue. Our results confirm that ZNF671 can be used as a biomarker, with low expression predicting worse outcomes in patients with BRCA, CESC, HNSC, KIRP, LUAD, PAAD, SARC, and UCEC.

AUTHOR CONTRIBUTIONS
JiZ and ZZ designed the research. JlZ, TX, YT, RL, BW, JL, and AX acquired and analyzed the data. JiZ, XH and YY wrote the manuscript.

FUNDING
This work was supported by grants from the Guangzhou Key Medical Discipline Construction Project Fund (B195002004042).