Orthodenticle Homeobox OTX1 Promotes Papillary Thyroid Carcinoma Progression and Is a Potential Prognostic Biomarker

Papillary thyroid carcinoma (PTC) is the most common type of thyroid neoplasms, characterized by evidence of follicular cell differentiation. Orthodenticle homeobox 1 (OTX1) is a transcription factor which has been implicated in numerous diseases, including malignancies. The objective of this research was to explore the function of OTX1 in PTC. Immunohistochemistry (IHC) was employed to determine the protein level of OTX1 in PTC specimens. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, a xenograft model on nude mice was established to investigate in vivo effects of OTX1. Our results revealed that OTX1 was significantly upregulated within specific PTC tissues and was remarkably correlated with unfavorable clinical outcomes in PTC. Silencing OTX1 resulted in a significant inhibition in cell viability and suppressed cell proliferation. In addition, in vivo experiments demonstrated that OTX1 silencing resulted in a significant suppression of tumor growth in nude mice. Collectively, these results suggest that OTX1 may play crucial roles in promoting PTC progression.


Introduction
Papillary thyroid carcinoma (PTC) represents the most prevalent type of thyroid cancers, accounting for about 80% of all thyroid malignancies [1].It is characterized by distinctive nuclear features, including nuclear pseudoinclusions and nuclear grooves [2].Despite being generally associated with a favorable prognosis, a subset of PTC cases exhibits aggressive behavior and increased risk of recurrence [3].Terefore, there is a critical need to identify molecular markers that can aid in predicting the prognosis and guiding treatment decisions for PTC patients.
Transcription factors are crucial regulators of gene expression that play pivotal roles in almost all cellular processes, including cellular proliferation, diferentiation, and survival [4].One such transcription factor is orthodenticle homeobox 1 (OTX1), which belongs to the homeobox gene family [5].Homeobox genes encode transcription factors categorized by a conserved DNA-binding domain known as the homeodomain, enabling them to bind to specifc DNA sequences and regulate expression of target genes [6].OTX1 is involved in important developmental processes, including embryonic development, neurogenesis, and organogenesis [7].It serves as a transcriptional regulator, infuencing the expression of genes necessary for proper tissue and organ formation during embryogenesis [8].However, aberrant expression of OTX1 has been observed in various cancers, such as lung cancer and colorectal cancer [9,10].In bladder cancer, OTX1 dysregulation has been linked to increased tumor cell proliferation and invasiveness [11].Similarly, in lung cancer, abnormal OTX1 expression has been associated with enhanced invasion capability of tumor cells, contributing to metastasis [12].In addition, an aberrant OTX1 level has been indicated in cervical cancer progression [13].
Despite these insights into OTX1's involvement in several cancers, its specifc role in PTC remains inadequately understood.PTC is the most common type of thyroid cancer characterized by diferentiated follicular cells.Given the importance of transcription factors in cellular processes and the emerging role of OTX1 in other cancers, investigating its role in PTC could provide valuable insights into the underlying molecular mechanisms driving PTC development and progression.
Terefore, the objective of the current study was to explore the expression pattern of OTX1 in PTC tissues and assess its correlations with clinicopathological characteristics.In addition, we aimed to examine the functional signifcance of OTX1 in PTC by investigating its impact on cell viability and growth.By conducting these investigations, we aimed to gain a deeper understanding of the roles of OTX1 in PTC and explore its clinical signifcance as a prognostic biomarker as well as a potential therapeutic target, specifically in the context of PTC.

Patient Samples and Immunohistochemistry (IHC)
Detection.A cohort of PTC patient samples (n � 294) was collected from Xi'an Gaoxin Hospital.Te inclusion criteria for the study encompassed histologically confrmed PTC cases in patients aged between 18 and 80 years, exclusively PTC cases without distant metastasis, and individuals who underwent surgical resection.Data were collected from PTC patients diagnosed within the period of 2010-2015, and only patients with a recorded survival month greater than zero were considered.IHC was performed to evaluate the expression of OTX1 in PTC tissues as previously reported using the antibody from Abcam (#ab25985; 1 : 200 dilution) [14].

Xenograft Model.
Nude mice were subcutaneously injected with PTC cells infected with OTX1-shRNA or negative control shRNA.Tumor growth was monitored every fve days, and tumor volume was calculated.After one month, xenografts were isolated for weighing.In total, 500,000 cells per 100 μL were seeded subcutaneously to start the xenograft experiment.

Statistical Analysis.
Descriptive statistics were used to describe patient characteristics such as age, sex, laterality, tumor diameter, diferentiation grade, T stage, N stage, surgery type, radiotherapy status, and OTX1 expression level.To assess the relationship between these variables and survival outcomes, Kaplan-Meier survival analyses were conducted, providing information on mean survival months, 5-year cancer-specifc survival (CSS) percentages, and corresponding p values.Te signifcance of the associations was evaluated using the chi-square test or Fisher's exact test for categorical variables and the t-test for cellular and animal experiments compared to the "scrambled-shRNA" group [17].SPSS and Prism software packages were employed in this study.
2.6.Ethics Approval.Tis study was conducted at Xi'an Gaoxin Hospital in compliance with the ethical guidelines and regulations set forth by the Institutional Review Board (IRB).Prior ethical approval was obtained to ensure the protection of human subjects and animal welfare.Informed consent was obtained from all participants, emphasizing voluntary participation, understanding of the study objectives and procedures, and the right to privacy and confdentiality.Patient ethics were upheld by ensuring anonymity, confdentiality, and the responsible use of collected data solely for research purposes.Animal ethics were strictly followed, adhering to the guidelines of the IACUC, ensuring appropriate housing, care, and handling.By obtaining ethical approval, obtaining informed consent, and prioritizing animal welfare, this study demonstrated a commitment to upholding ethical standards in research while safeguarding the rights and well-being of human participants and animal subjects.1 displays the comprehensive information of the 294 patients diagnosed with papillary thyroid carcinoma (PTC) included in this study.Te patients' characteristics are presented in terms of age, sex, laterality, tumor diameter, diferentiation grade, T stage, N stage, surgery type, and radiotherapy.Te cohort consisted of an equal distribution between patients younger than 49 years (50.0%,n � 147) and those aged 49 years or older (50.0%, n � 147).Te majority of patients were female (74.8%, n � 220) compared to males (25.2%, n � 74).Tumor distribution indicated 43.2% (n � 127) in the left lobe, 48.6% (n � 143) in the right lobe, and 8.2% (n � 24) in bilateral lobes.Tumor diameter was divided into <1.7 cm (50.0%, n � 147) and ≥1.7 cm (50.0%, n � 147).Diferentiation grade revealed that PTC cases were predominantly well diferentiated (78.9%, n � 232), with fewer cases classifed as moderately diferentiated (12.9%, n � 38) or poorly diferentiated (8.2%, n � 24).T-stage distribution showed T1 (50.7%, n � 149), T2 (18.7%, n � 55), T3 (24.8%,n � 73), and T4 (5.8%, n � 17).In terms of lymph node involvement (N stage), the majority were classifed as N0 (81.0%, n � 238), with smaller proportions of N1a (11.2%, n � 33) and N1b (7.8%, n � 23).Surgery types included lobectomy and/or isthmectomy (16.7%, n � 49), subtotal or near total thyroidectomy (3.4%, n � 10), and total thyroidectomy (79.9%, n � 235).Regarding radiotherapy, 57.5% (n � 169) of patients did not receive radiotherapy, while 42.5% (n � 125) accepted postoperative radiotherapy as part of their treatment.

3.2.
Expression of OTX1 in PTC Tissues.Immunohistochemical analysis revealed a high expression of OTX1 in 46.9% of PTC tissues (n � 138), whereas 81.0% of adjacent nontumor tissues (n � 238) exhibited weak or negative staining for OTX1 (Figure 1).Table 2 provides a comprehensive analysis of the correlation between OTX1 expression levels and various characteristics of the 294 PTC patients included in this study.No signifcant correlations were found between OTX1 expression and age or sex.Similarly, no signifcant association was detected between OTX1 levels and laterality or surgery type.However, a signifcant correlation was identifed between OTX1 expression and tumor diameter, with a higher expression in PTC patients with smaller tumor sizes (<1.7 cm, p � 0.010).Furthermore, OTX1 expression was marginally associated with the T stage, indicating a higher expression in advanced T stages (p � 0.051).Notably, a signifcant correlation was found between OTX1 expression and lymph node involvement (N stage), with a higher expression observed in patients with lymph node metastasis (p � 0.007).No signifcant correlations were observed between OTX1 expression and diferentiation grade or radiotherapy status.
Tese fndings indicated a critical role of OTX1 in PTC progression, particularly in tumor size determination and lymph node metastasis.Nevertheless, more investigations are essential to unravel underlying mechanisms and clinical implications of these associations.

OTX1 Indicates
Poor PTC Survival.Table 3 presents the results of Kaplan-Meier survival analyses conducted on a cohort of 294 patients with PTC.Te analysis revealed several signifcant associations between these variables and survival outcomes.Notably, younger patients (<49 years) had a signifcantly higher mean survival time (81.5 ± 0.5 months) and 5-year cancer-specifc survival (CSS) rate (99.3%) than older patients (≥49 years) (78.9 ± 1.4 months, 93.2% CSS, Figure 2(a)).In addition, tumor diameter was found to be signifcantly correlated with survival, with patients having tumors smaller than 1.7 cm showing a 100% 5-year CSS than those with larger tumors (92.3% CSS, Figure 2(b)).Other signifcant associations were observed between survival outcomes and the diferentiation grade, T stage, N stage, and OTX1 expression level.Welldiferentiated tumors had the highest 5-year CSS (99.5%), while poorly diferentiated tumors had the lowest (59.1%, Figure 2(c)).Similarly, patients with lower T (Figure 2(d)) and N stages (Figure 2(e)) exhibited better survival rates.Interestingly, a low OTX1 expression was associated with a higher 5-year CSS (97.9%) compared to high OTX1 expression (94.3%, Figure 2(f )).Te reason for missing survival times in certain subgroups (Table 3) is due to the 100% 5-year survival rate observed in these particular groups.As a result, the median survival time cannot be calculated for these specifc groups.Te 100% 5-year survival rate indicates that all patients in these groups survived for at least 5 years from the time of diagnosis, making the calculation of the median survival time impractical.Nevertheless, these fndings highlight the prognostic signifcance of age, tumor characteristics, and OTX1 expression in PTC, providing valuable insights for risk stratifcation and treatment decision-making in clinical practice.

OTX1 Silencing Inhibits Cell Viability and Growth.
We further provided compelling evidence regarding the impact of OTX1 on cell growth in PTC.MTT results clearly demonstrate a signifcant reduction in cell growth upon silencing OTX1 in both the BCPAP and TPC cell lines (Figures 3(a To corroborate the in vitro results, xenograft experiments were performed using nude mice to assess the efect of OTX1 silencing on tumor growth in a physiological setting.Strikingly, the xenografts derived from the OTX1-silenced BCPAP and TPC cell lines exhibited signifcantly slower growth rates than those from the control group (Figures 3(c) and 3(d)).Consistently, upon excising the xenografts, the OTX1-shRNA group exhibited a substantial reduction in tumor weight compared to the scrambled-shRNA group (Figures 3(e) and 3(f )).Tese fndings provide further Tese results unveiled the signifcance of OTX1 as a promising treatment target for interventions aimed at restraining the growth and progression of papillary thyroid carcinoma.Further investigations elucidating the underlying molecular mechanisms through which OTX1 infuences PTC development and progression are warranted to enhance our understanding of its functional signifcance in this malignancy.

Discussion
PTC is the most common type of thyroid neoplasm, characterized by its association with follicular cell diferentiation.Here, we aimed to explore the signifcance of orthodenticle homeobox 1 (OTX1), a member of the homeobox gene family and a transcription factor, in PTC progression and its potential as a prognostic biomarker.Our fndings contribute to the understanding of OTX1's involvement in PTC and align with the existing literature on the functional signifcance of transcription factors in cancer [18][19][20].
Transcription factors play pivotal roles in regulating gene expression and orchestrating various biological processes, including cellular proliferation, diferentiation, and survival.OTX1, a transcription factor, has been implicated in embryonic development, neurogenesis, and organogenesis [21].Moreover, its dysregulated expression has been discovered in several cancer types including ovarian cancer, prostate cancer, and colorectal cancer [9,22,23].Tese studies highlight the versatile role of OTX1 in diferent disease contexts, including cancer.
Immunohistochemistry (IHC) analysis of PTC tissues revealed a signifcantly higher OTX1 expression in certain tumor samples, indicating its potential as a prognostic biomarker in PTC.Tis fnding is consistent with previous data demonstrating the clinical relevance of OTX1 expression in cancer.For instance, research conducted on hepatocellular carcinoma has shown that an elevated OTX1 expression is associated with a poor prognosis and tumor progression [24].In addition, in lung cancer, OTX1 overexpression has been linked to advanced disease stage and

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Genetics Research reduced survival [10].Collectively, these fndings underscore the signifcance of OTX1 as a potential prognostic indicator in various cancer types.
In vitro experiments using PTC cell lines further elucidated the functional role of OTX1 in PTC progression.OTX1 silencing resulted in reduced cell viability and inhibited cell growth, suggesting its crucial involvement in promoting PTC cell proliferation.Tese fndings align with those of previous studies on OTX1 in cancer.In lung cancer, OTX1 knockdown has been shown to suppress cell growth and induce apoptosis, highlighting its potential as a therapeutic target [10].Similarly, in laryngeal squamous cell carcinoma, targeting OTX1 impairs tumor cell proliferation and tumor growth [16].Tese studies provide additional support for the functional signifcance of OTX1 in cancer cell biology.
In vivo xenograft experiments using nude mice corroborated the in vitro fndings, demonstrating that silencing OTX1 signifcantly suppressed tumor growth in subcutaneous PTC xenografts derived from BCPAP and TPC cell lines.Tis observation is in line with that of previous studies implicating OTX1 in tumor progression.In esophageal squamous cell carcinoma, overexpressing OTX1 has been shown to promote tumor growth as well as invasion in nude mice [25].Moreover, in pancreatic cancer, targeting OTX1 has been associated with reduced tumor growth and metastasis [26].Tese studies underscore the importance of targeting OTX1 as a therapeutic direction to inhibit tumor progression in various cancer types.
In addition, the Kaplan-Meier survival analyses demonstrated a signifcant association between high OTX1 expression and reduced cancer-specifc survival in PTC cases.Tis fnding is consistent with previous results indicating the clinical relevance of OTX1 in cancer prognosis.In gastric cancer, an elevated OTX1 expression has been correlated with worse survival [19].Similarly, in ovarian cancer, a high OTX1 level has been associated with advanced disease stage and unfavorable prognosis [23].Tese studies collectively emphasize the signifcance of OTX1 as a prognostic marker across diferent cancer types.
Tis study signifcantly advances our understanding of OTX1's role in thyroid cancer, particularly papillary thyroid     Student's t-test was conducted, and statistical signifcance was denoted as * p < 0.05.

Conclusion
Our study provides evidence for the oncogenic role of OTX1 in papillary thyroid carcinoma (PTC).Te high expression of OTX1 in PTC tissues is signifcantly associated with a worse prognosis.Moreover, OTX1 silencing impairs cell viability and growth both in vitro and in vivo.Our fndings suggest that OTX1 may serve as a potential prognostic biomarker and therapeutic target for PTC.Further studies are needed to elucidate the underlying molecular mechanisms and explore the clinical implications of targeting OTX1 in PTC management.
) and 3(b)).Tese observations highlight the crucial role of OTX1 in promoting the proliferation of PTC cells.

Figure 3 :
Figure 3: Efects of OTX1 silencing on cell growth and tumor progression in papillary thyroid carcinoma (PTC).(a, b) Cell viability assays revealed a signifcant reduction in cell growth following OTX1 silencing in both BCPAP and TPC1 PTC cell lines.(c, d) In vivo xenograft experiments using nude mice demonstrated slower subcutaneous tumor growth upon OTX1 silencing, with tumors generated from BCPAP and TPC1 cell lines, respectively.(e, f ) Analysis of excised xenografts showed a notable decrease in tumor weight in the OTX1-shRNA group compared to that in the scrambled-shRNA group.Tese fndings provide compelling evidence for the crucial role of OTX1 in promoting cell growth and tumor progression in PTC, supporting its potential as a therapeutic target.Te error bars represent the standard deviation.Student's t-test was conducted, and statistical signifcance was denoted as * p < 0.05.

Table 2 :
Correlations between OTX1 expression and PTC patients' characteristics.

Table 3 :
Kaplan-Meier survival analyses of the enrolled PTC cohort.