Abstract
Background/Aim: Zinc finger CCCH-type containing 12A (ZC3H12A), also known as monocyte chemotactic protein-induced protein 1, has emerged as having a potential role in the landscape of some human cancer types, contributing to anti-tumorigenesis through signaling pathways of inflammation, apoptosis, autophagy and angiogenesis. However, its specific impact on the prognosis of oral squamous cell carcinoma (OSCC) remains to be investigated.
Patients and Methods: We conducted in-vitro cell models in a pilot study and performed a retrospective cohort study involving 242 patients with OSCC to unravel the association between ZC3H12A expression level and oral cancer prognosis during 3, 5, and 10-year follow-up.
Results: The findings showed that endogenous ZC3H12A expression was decreased in both the malignant (BQO) and highly metastatic (HSC-3-M3) OSCC cell lines compared to dysplastic oral keratinocytes (DOK) and the parental cell line of HSC-3-M3 (HSC-3). The analysis of clinical cancerous tissue arrays from patients with OSCC showed a significant association between complete loss of ZC3H12A expression and heightened cancer mortality, particularly at 3 and 5 years. Moreover, the complete loss of ZC3H12A expression may be associated with increased risk of lymph node involvement in OSCC.
Conclusion: Our investigation suggests that the complete loss of ZC3H12A expression exacerbated the unfavorable prognosis of OSCC in this Taiwanese population, but further study on elucidating the gradual decline of ZC3H12A expression in OSCC is imperative.
Introduction
Global Cancer Statistics 2025 estimates that the United States will experience approximately 2,041,910 new cancer cases and 618,120 cancer-related deaths (1). The age-adjusted incidence rate remains steadfast, hovering around 22.8% per 100,000 persons for cancer of the oral cavity, oropharynx and hypopharynx (ICD-O-3: C00-C06, C09-C10, C12-14) from 2011 to 2020. Davaatsend et al. reported a 5-year survival rate of 50.3% for patients with oral cancer and 38% for those with tongue cancer within a Mongolian population (2). In contrast, a cohort study involving 14,059 patients from 10 population-based cancer registries in India showed an overall 5-year survival rate of 37.2% for patients with oral cancer (3). Surgery, radiotherapy, and chemotherapy are still the standard medical procedures for oral cancer. The willingness of patients with oral cancer to try other auxiliary treatments, such as targeted therapy and immunotherapy, which often come with self-pay support, is increasing, and the research and development of potential therapeutic biomarkers to improve prognosis remains necessary.
The common risk factors involved in the initiation of oral cancer include tobacco and betel quid chewing, cigarette smoking, alcohol consumption, and infection with human papillomavirus types 16 and 18. Betel quid and areca nut chewing were evaluated as Group 1 carcinogens to humans by the International Agency for Research on Cancer in 2014 (4). Of particular note is the formidable impact of betel chewing on potentially malignant oral lesions in Taiwan, particularly in cases of oral leukoplakia and oral submucosal fibrosis (5, 6). Moreover, it is the major risk factor for developing oral squamous cell carcinoma (OSCC) in Taiwan. OSCC is a common malignant neoplasm for men in Taiwan, and OSCC metastasizing to regional lymph nodes significantly reduces prognosis and survival (7, 8).
The inflammatory response of the nuclear factor-kappa B (NF-κB)–cyclooxygenase-2 (COX2) axis contributes to diverse stages of cancer initiation, promotion, and progression (9, 10). Overexpression of COX2 level is involved in oral carcinogenesis and cervical node metastases (11-13). The effect of areca nut extract on the up-regulation of inflammatory signaling within the NF-κB–COX2 axis has been studied on human oral keratinocytes and OSCC cell lines (14, 15), and prolonged inflammation is associated with oral cancer development and progression (16, 17). Our previous study showed that a selective COX2 inhibitor can provide a preventative effect against cancer occurrence and progression of OSCC (18). Beyond the extrinsic inflammation triggered by betel chewing, out-of-control intrinsic inflammation of the tumor site can result in immunosuppression, thereby providing a preferred tumor microenvironment for the progression of OSCC.
Zinc finger CCCH-type containing 12A (ZC3H12A), also known as monocyte chemotactic protein-1-induced protein-1 (MCPIP1), controls immune responses by regulating cytoplasmic mRNA decay (19). In a Zc3h12a-knockout mouse model, Zc3h12a destabilized interleukin (IL)-6 mRNA and IL-17 activity, effectively inhibiting IL-17-mediated signaling and reducing inflammation (20). ZC3H12A was shown to be essential for mediating self-inflicted DNA breaks following DNA damage in small-cell lung cancer cells (21). Overexpression of ZC3H12A inhibited metastasis and tumor growth in breast cancer by downregulating the expression of the IL-17 signaling pathway-related proteins, including IL-17 receptor A, IL-17A, and NF-κB activator 1 (22). In patients’ outcomes from the Human Protein Atlas database, however, a low expression of ZC3H12A appears to be favorable for survival in renal cell carcinoma, and the 5-year survival rate in patients with low and high expression levels were 78% and 63%, respectively (https://www.proteinatlas.org/ENSG00000163874-ZC3H12A/pathology). These inconsistent findings might result from genetic diversity or different environmental exposures, but this remains to be elucidated.
RNA-Seq data from the The Cancer Genome Atlas (TGCA) dataset show that the ZC3H12A gene expression level varies in human cancer tissues, and it is highly expressed in patients with head and neck cancer (n=499; median fragments per kilobase per million=20.3) compared with other cancer types. In this study, we attempted to investigate the distribution of ZC3H12A expression in cancerous tissues from patients with OSCC and to elucidate the potential effect of ZC3H12A expression in the prognosis and survival of patients with betel-related OSCC in Taiwan.
Patients and Methods
Database patients and methods. This research received ethical approval from the Ethics Committee of Changhua Christian Hospital, Taiwan. All patients provided written informed consent. The study was conducted in accordance with the guidelines of the Institutional Review Board, under approval number 200207, and adhered to the principles outlined in the Declaration of Helsinki.
We obtained sex, pathological stage, and ZC3H12A RNA-Seq data (fragments per kilobase per million) from 431 patients with head and neck squamous cell carcinoma (HNSCC) in the database of TCGA for pilot analysis. Gene expression profiles of HSC-3 and HSC-3-M3 cells by Sakurai et al. (23) were downloaded from the Gene Expression Omnibus (GSE160395). The normalized ZC3H12A signal was extracted from quadruplicate SurePrint G3 Human v3 microarray data (Agilent Technologies, Santa Clara, CA, USA).
In-vitro cell models. A dysplastic oral mucosa cells cell line (DOK), derived from a 57-year-old male with oral epithelial dysplasia (24), was purchased from the European Collection of Authenticated Cell Cultures (ECACC #94122104; Porton Down, Salisbury, UK). BQO cell line originated from a 55-year-old patient with OSCC (stage IVa, T2N2M0) with a history of betel nut chewing but not of alcohol use or smoking (18). HSC-3 and HSC-3-M3 cells (JCRB0623, JCRB1354) were obtained from the Japanese Cancer Research Resources Bank (Ibaraki City, Osaka, Japan) and were derived from the same patient with tongue cancer, with HSC-3-M3 exhibiting higher metastatic capacity (25). DOK was cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM L-glutamine, 5 μg/ml hydrocortisone, 10% fetal bovine serum, and antibiotics. OSCC cell lines were maintained in DMEM/F-12 with L-glutamine, HEPES, sodium pyruvate, sodium bicarbonate, hydrocortisone, FBS, and antibiotics (Gibco, Invitrogen, Grand Island, NY, USA).
Western blotting of cell lines. Total protein was extracted frm cells using RIPA lysis buffer and quantified by a bicinchoninic acid assay. Protein samples (30 μg) underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bolt Mini Gel Tank; Invitrogen). In accordance with standard laboratory practices, the expression levels of all target proteins within the same signaling pathway were analyzed using a single polyvinylidene difluoride membrane. This approach ensures consistency and enables reliable comparison of differential protein expression. The membrane was sectioned based on the molecular weight markers indicated by a prestained protein ladder, allowing for precise isolation and subsequent hybridization of specific membrane regions corresponding to each target protein. Primary antibodies included those against ZC3H12A (GeneTex, Irvine, CA, USA); focal adhesion kinase (FAK), twist-related protein 1 (TWIST1), snail family transcriptional repressor 1 (SNAI1), MYC proto-oncogene bHLH transcription factor (MYC), glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA, USA); and actin (Sigma-Aldrich, St. Louis, MO, USA) (all diluted 1:1,000). Secondary antibodies were goat anti-mouse (Merck Millipore, St. Louis, MO, USA) and anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA) (both diluted 1:10,000). Chemiluminescent signals were captured using ImageQuant LAS 4000 (ImageQuant LAS 4000; GE Healthcare, Pittsburgh, PA, USA).
Immunohistochemistry of clinical samples. Tissue microarrays (TMAs) were constructed from samples from patients with oral cancer collected between 2000 and 2019 at Changhua Christian Hospital, Taiwan, ROC. Recurrent cases and small samples (<0.1 cm) were excluded, yielding a total of 242 OSCC cases (232 males, 10 females). Cancer staging followed the American Joint Committee on Cancer TNM system (26). Pathologists reviewed hematoxylin and eosin-stained TMAs to confirm morphology and grade. TMAs were dewaxed, rehydrated, and treated with 3% H2O2 in methanol. After ethanol and phosphate-buffered saline hydration, slides were incubated in citrate buffer (pH 6.0), followed by ZC3H12A primary antibody (1:200, GeneTex) for 20 min. After phosphate-buffered saline washes, peroxidase-labeled secondary antibody was applied for 30 min, followed by diaminobenzidine and hematoxylin staining. Positive and negative controls ensured reliability. Two pathologists independently scored immunostaining intensity as negative, weakly positive (1+), or moderately-to-strongly positive (2+) (Figure 1).
Zinc finger CCCH-type containing 12A (ZC3H12A) expression from The Cancer Genome Atlas dataset and in-vitro cell models. In the pilot study, we analyzed (A) RNA-Seq data of ZC3H12A from 431 patients with head and neck squamous cell carcinoma; (B) ZC3H12A protein expression level in oral pre-cancerous cells [dysplastic oral keratinocytes (DOK)] and betel-related oral squamous cell carcinoma cells (BQO); and (C) endogenous protein levels of ZC3H12A, focal adhesion kinase (FAK), twist-related protein 1 (TWIST1), snail family transcriptional repressor 1 (SNAI1), and MYC proto-oncogene bHLH transcription factor (MYC) in HSC-3 and HSC-3-M3 OSCC cells. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase (D) Relative quantification of endogenous mRNA and protein levels of ZC3H12A in HSC-3 and HSC-3-M3 cells. Data are the mean±standard error of the mean. All data represent the results of at least three independent experiments, each conducted in triplicate. FPKM: Fragments per kilobase per million. All statistical analyses were performed using unpaired t-tests, with p<0.05 indicating statistical significance.
Statistical analysis. Standard deviation and standard error of the mean were used for reporting the expression levels of ZC3H12A in TCGA-HNSCC data and OSCC cell lines (Figure 2). Associations between clinical characteristics, mortality, and ZC3H12A expression in patients with OSCC were assessed using Student’s t-test and chi-square test over 3-, 5-, and 10-year follow-up. Multiple logistic regression models evaluated the relationship between ZC3H12A expression in cancerous tissues and OSCC mortality risk, reporting crude and adjusted odds ratios (aOR) with 95% confidence intervals (CI) and p-values. Cox proportional hazards regression estimated crude and adjusted hazard ratios (aHR) for mortality risk over time. Follow-up (in months) was measured from the start of standard OSCC treatment (per CCH Oral Cancer Guideline) to death or the last visit (27). Kaplan-Meier curves and log-rank tests were used to evaluate survival outcomes based on baseline ZC3H12A expression. Binary and ordinal logistic regression models examined the association between ZC3H12A expression andrisk of lymph node involvement. All analyses were conducted using SAS 9.4, with values of p≤0.05 being considered statistically significant.
Immunohistochemical staining and scoring of zinc finger CCCH-type containing 12A (ZC3H12A) expression in tissue microarrays from patients with oral squamous cell carcinoma. Immunohistochemistry-stained specimens were scored under a microscope at magnifications of 100× and 200× by pathologists. A semi-quantitative scoring of the intensity of tumor cytoplasmic staining of ZC3H12A was recorded for all patients.
Results
A pilot study of ZC3H12A expression in patients with HNSCC and oral cell models. From data of 431 patients with HNSCC in the TGCA dataset, reduced mRNA expression of ZC3H12A was present in patients with late cancer stages (III and IV) compared to those with early stage (I and II) disease (Figure 2A). After sex stratification, this reduced ZC3H12A mRNA level was explicitly observed in male patients with HNSCC. However, this did not reach statistical significance.
In cell models, we observed a significantly reduced level of endogenous ZC3H12A in BQO cells compared with DOK (Figure 2B). HSC-3 and HSC-3-M3 cell lines were used to test the endogenous protein level of epithelial–mesenchymal transition (EMT) markers (FAK, TWIST1, SNAI1, and MYC) and ZC3H12A (Figure 2C). HSC-3-M3 showed a higher expression of MYC than HSC-3. In contrast, no difference was observed in the selected markers related to EMT. Regarding the endogenous level of ZC3H12A, we found that HSC-3-M3 showed a lower protein expression (75.8%) than HSC-3. This result corresponded to the mRNA expression levels of ZC3H12A in both cell lines from the Gene Expression Omnibus dataset (GSE160395) (Figure 2D).
Association of clinical characteristics, ZC3H12A expression, and mortality in patients. In the study population, 16.9% of patients with early stage (I and II) and 43.8% of patients with late stage (III and IV) disease had radiotherapy, while 6.6% and 17.8% of patients, respectively, had chemotherapy. In addition, 10 patients (4.1%) had missing radiotherapy and chemotherapy medical records. The clinical characteristics relative to mortality in 3-, 5-, and 10-year follow-up are shown in Table I.
Clinical characteristics relative to cancer mortality in patients with oral squamous cell carcinoma (n=242).
In the study population, cancer stage, tumor size, regional lymph node involvement, pathological grade, and treatment with radiotherapy or chemotherapy were significantly associated with cancer mortality in patients with OSCC, as were common clinical risk factors in human cancer.
The expression of ZC3H12A by immunohistochemistry in TMAs from 242 patients with OSCC were presented as negative, weak, moderate, or strong (Figure 1) in 84, 107, 43, and 8 patients, respectively. These data were then analyzed in dichotomous and trichotomous scoring models (Table II). The analysis revealed a significant association between cancer mortality and ZC3H12A expression level under the dichotomous scoring model (negative vs. positive) in the 3-year and 5-year follow-up (p=0.018 and p=0.030, respectively). However, the association with cancer mortality was only significant at the 3-year follow-up (p=0.046) under the trichotomous scoring model (negative, weakly positive, and moderately-to-strongly positive).
Association between immunohistochemical (IHC) expression of zinc finger CCCH-type containing 12A (ZC3H12A), clinical characteristics, and cancer mortality in patients with oral squamous cell carcinoma (n=242).
Remarkably, we observed a significant relationship between late cancer stage and negative ZC3H12A expression in the dichotomous scoring model (p=0.008 and p=0.022, respectively). Cancer stage classification is based on the pathological TNM staging system, comprised of the primary tumor (T), regional lymph nodes (N), and distant metastasis (M). Patients with OSCC with positive ZC3H12A expression in cancerous tissues had a significantly lower frequency of regional lymph node involvement (N1) in both scoring models (p=0.002 and p=0.001, respectively). Following stratification by the primary tumor size (T), it became evident that the ZC3H12A scoring level exhibited a robust association with regional lymph node involvement (N1) among patients with T1 or T2 stage disease (p=0.0009 and p=0.0012, respectively). However, no such correlation was observed in patients with T3 or T4 stage. Additionally, patients displaying positive ZC3H12A expression demonstrated a lower incidence of regional lymph node involvement in those with T1 or T2 stage. No significant association was shown between the pathological grading and ZC3H12A scoring in patients with OSCC.
Loss of ZC3H12A expression increased the risk of cancer mortality. After adjusting for potential confounders, patients with negative ZC3H12A expression showed a significantly increased overall mortality risk at 3 years [adjusted hazard ratio (aHR)=1.65, 95% CI=1.10-2.48] and 5 years (aHR=1.48, 95% CI=1.01-2.16) comparing with those with positive expression (Table III). There was no increased risk of cancer mortality among patients exhibiting negative expression of ZC3H12A in the 10-year follow-up. The analysis of cancer mortality risk associated with ZC3H12A expression levels using multiple regression models is shown in Table IV.
Risk of cancer mortality due to suppression of zinc finger CCCH-type containing 12A (ZC3H12A) expression as determined by immunohistochemistry in patients with oral squamous cell carcinoma during different follow-up periods.
Risk of cancer mortality due to suppression of zinc finger CCCH-type containing 12A (ZC3H12A) expression as determined by immunohistochemistry in patients with oral squamous cell carcinoma during different follow-up periods.
Figure 3 illustrates the estimated disease-free survival rates among patients with OSCC with negative and positive (weak and moderate-to-strong) expression of ZC3H12A at the 3-, 5-, and 10-year follow-up. Statistical significance between the survival curves was assessed using the log-rank test, revealing that the survival curve for patients with negative ZC3H12A expression differed significantly from those with positive expression in both the 3-year and 5-year follow-up (p=0.019 and p=0.022, respectively) in the dichotomous scoring model. Over the 10-year follow-up, we did not observe a significant association between ZC3H12A expression and cancer mortality. This outcome may have been influenced by multiple factors, such as disease progression, tumor phenotype changes, treatment effects, protein expression alterations, comorbidities, and individual patient characteristics. The survival curve analysis in the trichotomous scoring model showed non statistically significantly differences between expression levels in follow-up after adjustment with the Sidak correction (p*).
Disease-free survival curves for patients with oral squamous cell carcinoma according to dichotomous (A) and trichotomous (B) scoring of zinc finger CCCH-type containing 12A (ZC3H12A) expression by immunohistochemistry. p*: The p-value from the multiple comparisons for the log-rank test with Sidak correction.
Loss of ZC3H12A expression increased the risk of lymph node metastasis. The outcomes of binary logistic and cumulative logistic regression models for investigating the impact of a negative ZC3H12A expression in OSCC cancerous tissue on the risk of lymph node involvement are shown in Table V. After adjusting for age, sex, histological differentiation, and treatments with radiotherapy and chemotherapy, both regression models revealed a significant association between loss of ZC3H12A expression and lymph node metastasis among OSCC patients in the dichotomous scoring model (p=0.0030 and p=0.0077, respectively). The risk was particularly pronounced at more than 4-fold when compared with patients exhibiting moderately-to-strongly positive expression of ZC3H12A in the trichotomous scoring analysis (p=0.0040 and p=0.0287, respectively). These findings suggest that the loss of ZC3H12A expression in cancerous tissues may contribute to regional lymphatic metastasis of OSCC.
Association of reduced zinc finger CCCH-type containing 12A (ZC3H12A) expression with the risk of lymph node involvement in patients with oral squamous cell carcinoma.
Discussion
ZC3H12A, also known as MCPIP1, is a zinc finger endoribonuclease that plays a pivotal role in regulating both adaptive and innate immune responses as well as inflammation (28, 29). It is induced by several inflammation-related stimuli, including lipopolysaccharide, IL-1β, and tumor necrosis factor-alpha (TNF-α). As a key regulator of the inflammatory response, ZC3H12A modulates inflammation through multiple mechanisms. In the TNF-α, lipopolysaccharide, and IL-1β-mediated NF-κB and c-Jun N-terminal kinase signaling pathways, ZC3H12A functions as a deubiquitinase, removing ubiquitin chains from key signaling proteins such as TNF receptor associated factor 2 (TRAF2) and TRAF3. This action disrupts the activation of the NF-κB pathway, thereby suppressing inflammation (30). However, ZC3H12A exhibits ribonucleolytic activity and promotes the degradation of mRNAs encoding inflammatory mediators, such as IL-1β, IL-6, IL-12β, IL-2, REL proto-oncogene, and TNF receptor superfamily member 4 by cleaving their 3′ untranslated regions, thereby contributing to the post-transcriptional regulation of inflammation (31).
Reduced mRNA expression of ZC3H12A was observed in patients with late-stage HNSCC from the TGCA database, although this was not a significant difference. This study showed that the frequency of patients with late stage OSCC (III and IV) presenting a complete loss of ZC3H12A expression in the cancerous tissues was higher compared to those with early-stage disease (I and II). Remarkably, patients with complete loss of ZC3H12A expression showed increased cancer mortality and reduced relapse-free survival rate, and the poor prognosis in these patients may be associated with N1 lymph node metastasis. However, the unequal representation of males and females in this study, with 232 males and only 10 females in the clinical cohort, introduces a potential sex bias. While this imbalance limits the ability to perform a robust sex-based analysis, the primary focus of our study was on the overall prognostic significance of ZC3H12A in OSCC, a condition that is more prevalent in males, particularly in betel nut-related cases in Taiwan. To address this limitation, we suggest future studies with larger and more balanced cohorts are necessary to better understand the potential influence of sex on ZC3H12A expression and OSCC outcomes.
ZC3H12A expression has been linked to tumor staging in colorectal cancer and disease progression in breast cancer (22, 31). It can promote cancer cell apoptosis by inhibiting anti-apoptotic genes (32), and it acts as a transcription factor in the monocyte chemotactic protein-1-induced angiogenesis (33). Additionally, ZC3H12A plays a role in the metastasis of renal cell carcinoma by regulating EMT (34).
The etiopathogenesis of reduced ZC3H12A expression during progression of OSCC remains unclear. In the database of Catalogue Of Somatic Mutations In Cancer (GRCh38, COSMIC v98, https://cancer.sanger.ac.uk/cosmic), only a 1.72% (23/1340 tested SCCs) rate of somatic mutation was observed in patients with upper aerodigestive tract cancer after excluding one patient with nasopharyngeal carcinoma; the hotspot mutation was located at ZC3H12A c.925+71C>G. No hypomethylation of the ZC3H12A gene was found in the 496 tested samples. The molecular mechanism that leads to downregulation or suppression of ZC3H12A expression in cancerous tissues needs further investigation.
Although aberrant activation of EMT signaling during the invasion-metastasis cascade has been widely accepted (35, 36), only 0.01% of cancer cells are estimated to enter the systemic circulation for the formation of secondary tumors (37). However, several studies have raised concerns about the contribution of EMT to escaping chemotherapy, hypoxia, and other effects rather than metastasis formation (38, 39). We did not observe a difference in the protein expression levels of FAK, TWIST1, and SNAI1 between HSC-3 and HSC-3-M3 cells. Moreover, no EMT-activating transcription factor was highly altered in the mRNA expression profile of HSC-3-M3 cells, including SNAI1, SNAI2 (SLUG), TWIST1, zinc-finger E-box-binding homeobox 1 (ZEB1) and ZEB2 (23). However, significant up-regulation of NF-κB, COX2, and MYC expression was found in HSC-3-M3 calls. IκB kinase α, one of the primary activators of the inflammatory transcription factor NF-κB, can stabilize MYC by phosphorylating it on serines-67/71. This may lead to increased transcriptional activity of MYC resulting in higher proliferation and reduced apoptosis in carcinogenesis (40). Overexpression of NF-κB and COX2 inflammatory signaling is commonly observed in Taiwanese patients with OSCC due to long-term betel chewing (15), and NF-κB activation contributes to lymphatic metastasis of OSCC (41). In normal cells, the up-regulation of ZC3H12A can be initiated in response to genotoxic NF-κB signaling (42), and ZC3H12A can form a complex with TRAF family member-associated NF-kappa-B activator (TANK) and a USP10 deubiquitinase for the resolution of genotoxic NF-κB activation upon DNA damage (43).
The malignant transformation of oral submucous fibrosis into OSCC is approximately 3.7%, in a mean duration of 3.1 years in Taiwanese patients (44). Arecoline, the principal alkaloid of areca nut, causes cytotoxicity and inflammatory response in normal human gingival fibroblasts (45) and leads to the development of oral submucosal fibrosis (4). The most prominent non-immune cell types in the tumor microenvironment of OSCC are cancer-associated fibroblasts, which secrete several proinflammatory mediators (46, 47) and promote OSCC cell growth and invasion (48-50). Meanwhile, the expression level of pro-IL-1β was shown to be significantly raised from normal oral cells to dysplasia and OSCC during oral malignant transformation. The level of IL-1β secreted in the human OSCC cell lines was increased compared with DOK (51).
The ZC3H12A gene contains four functional NF-κB-binding sites, and ZC3H12A endoribonuclease acts as a negative regulator by promoting the degradation of active cytokine-induced inflammatory mRNAs on the NF-κB-dependent pathway during the early phase of inflammation, which is induced by TNF-α and IL-1β (33, 52). In addition, ZC3H12A can strongly inhibit the promoter of lipocalin 2, which forms a complex with matrix metalloproteinase-9 to increase its stability. This matrix metallopeptidase contributes to the tumor microenvironment, cancer cell invasion, and metastasis by regulating the mRNA stability of NFKBIZ, which plays a role in inflammatory responses by interaction with NF-κB proteins through ankyrin-repeat domains (53).
Conclusion
ZC3H12A can reduce NF-κB activity and act as a negative regulator in control of locoregional inflammation and in the tumor microenvironment (54). The complete loss of ZC3H12A in OSCC tissues may permit cancer cells to acquire an invasive phenotype, pushing them towards a multistep metastatic cascade via inflammatory deterioration. This leads to a poor prognosis in patients with betel-related OSCC.
Acknowledgements
This work was funded by the Changhua Christian Hospital (109-CCH-PRJ-002), I-Shou University (ISU-111-01-12A), and partially supported by the Ministry of Science and Technology (MOST 106-2314-B-039-024-MY2), Taiwan.
Footnotes
Authors’ Contributions
SLC: Writing–original draft, visualization, validation, methodology, investigation, formal analysis, data curation, conceptualization. JJT: Writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation, conceptualization. YNH: Writing–original draft, visualization, validation, methodology, investigation, formal analysis, data curation, conceptualization. JWL: Writing – original draft, visualization, validation, methodology, investigation, formal analysis, data curation, and conceptualization. SYH: Validation, methodology, investigation, and formal analysis. YJT: Validation, methodology, investigation, formal analysis. SHL: Writing – review and editing, supervision, resources, project administration, methodology, and conceptualization. All Authors read and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare there are no potential conflicts of interest.
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.
- Received July 25, 2025.
- Revision received September 6, 2025.
- Accepted September 10, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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