Objective: To explore the relationship between preoperative thyroglobulin (Tg) levels and recurrence risk as well as treatment response in patients with differentiated thyroid cancer (DTC). Methods: A retrospective analysis was conducted on the clinical data of 604 patients with DTC who underwent total thyroidectomy at Shanghai Renji Hospital from January 1, 2020, to December 31, 2024. The postoperative recurrence status of patients was recorded, with follow-up ending on June 30, 2025. Determine the cut-off value of preoperative Tg for predicting patient prognosis using the maximum selected rank statistic method. Multivariate Cox proportional hazards regression model was used to analyze the risk factors for postoperative recurrence in DTC patients. Survival curves were plotted using the Kaplan-Meier method, and the log-rank test was used to evaluate the differences in postoperative disease-free survival rates and treatment response among DTC patients with different preoperative Tg levels. Results: Among the 604 patients, 417 were female and 187 were male, with the age of 45.0(36.0, 57.0) years. The preoperative Tg was 20.2 (10.4, 44.3) μg/L, and the follow-up period was 1.40 (0.85, 2.84) years. Multivariate Cox proportional hazards regression model analysis revealed that preoperative Tg levels≥36.2 μg/L (HR=14.180, 95%CI:1.745-115.200) was a risk factor for postoperative recurrence in DTC. Patients with the preoperative Tg level of<36.2 μg/L had a higher 3-year disease-free survival rate compared to those with the preoperative Tg level of≥36.2 μg/L (99.8% vs 92.3%, P<0.001). The proportion of patients with the favorable treatment response was lower among those with the preoperative Tg level of≥36.2 μg/L compared to those with the preoperative Tg level of<36.2 μg/L, whereas the proportions of patients with biochemical incomplete, structural incomplete, and indeterminate responses were higher among those with the preoperative Tg level of≥36.2 μg/L (all P<0.05). Conclusions: Preoperative Tg levels are significantly associated with the recurrence risk and treatment response in DTC patients. The Tg level of≥36.2 μg/mL indicates a high recurrence risk and poor treatment response.

Objective: To analyze the risk factors for differentiated thyroid cancer(DTC) with distant metastases and establish a suitable prognostic risk prediction model. Methods: A retrospective analysis was performed on the clinical data of 2 337 patients diagnosed with distant metastasis of DTC from January 2000 to December 2021, as recorded in the Surveillance, Epidemiology, and End Results (SEER) database. Using the sample function in R, patients were randomly divided into a training set (n=1 635) and an internal validation set (n=702) at a ratio of 7∶3. Additionally, 227 patients with DTC and distant metastasis from Tianjin Medical University Cancer Institute and Hospital between January 2010 and December 2021 were included as an external validation set. Univariate and multivariate Cox proportional hazards regression model analysis, least absolute shrinkage and selection operator (LASSO) regression analysis were employed to screen for prognostic factors affecting the outcomes of patients with DTC and distant metastasis, and a prognostic prediction model nomogram was constructed. The predictive ability, accuracy, and clinical applicability of the model were evaluated using the area under the receiver operating characteristic curve (AUC), calibration curves, and decision curve analysis (DCA). Results: A total of 2 337 patients with distant metastasis of DTC were retrieved from the SEER database, including 988 males and 1 349 females, with the age of (58.4±18.6) years. Among them, 1 254 patients died with a median overall survival (OS) time of [M (Q1, Q3)] 42.0 (13.0, 95.0) months. At Tianjin Medical University Cancer Institute and Hospital, a total of 227 patients with DTC accompanied by distant metastasis were included, comprising 62 males and 165 females, with the age of (56.7±16.8) years. Among these patients, 114 died with a median OS time of 43.0 (15.0, 101.0) months. The follow-up ended upon the patient's death or March 31, 2025. After analysis using a univariate Cox proportional hazards regression model and screening via LASSO regression, 4 variables were included from the training set, including age, maximum tumor diameter, invasion of vital tissues, and surgical treatment. Multivariate Cox proportional hazards regression model analysis showed that age≥55 years (HR=3.37, 95%CI: 3.08-4.53), maximum tumor diameter>4 cm (HR=1.83, 95%CI: 1.12-2.99), and invasion of vital tissues (HR=2.15, 95%CI: 1.81-2.56) were risk factors affecting the OS of patients with DTC accompanied by distant metastasis, while surgical treatment (HR=0.44, 95%CI: 0.36-0.53) was a protective factor. A prognostic prediction model for patients with DTC accompanied by distant metastasis was constructed using these influencing factors. The AUC for predicting 3, 5, and 10 year OS were 0.780 (95%CI: 0.754-0.805), 0.769 (95%CI: 0.743-0.795), and 0.836 (95%CI: 0.809-0.864)(in the training set); 0.804 (95%CI: 0.768-0.841), 0.815 (95%CI: 0.779-0.850), and 0.877 (95%CI: 0.844-0.911)(in the internal validation set); and 0.753 (95%CI: 0.682-0.824), 0.717 (95%CI: 0.642-0.792), and 0.810 (95%CI: 0.725-0.894)(in the external validation set). The calibration curves demonstrated good fit between the predicted and observed values (all P>0.05), and DCA indicated that the model had high clinical application value. Conclusions: Age≥55 years, maximum tumor diameter>4 cm, and vital tissue invasion are independent risk factors for poor survival in patients with metastatic DTC, whereas surgical treatment improves prognosis. The proposed nomogram provides reliable prognostic assessment and may support individualized risk stratification and clinical decision-making.

Objective: To investigate the clinicopathological features of primary pulmonary epithelioid hemangioendothelioma (PEHE). Method: Forty cases of PEHE were diagnosed from October 2010 to June 2024 at the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. A retrospective analysis was conducted on their histological features, imaging findings, immunohistochemical characteristics and molecular phenotypes. Subsequently, the clinicopathological features were summarized. The patients were followed up. Result: Of the 40 cases, there were 19 males and 21 females, age 52.5 (43.0, 62.0) years old. Most patients were admitted for respiratory symptoms, mainly cough (25/40) and expectoration (14/40). Computed tomography findings mainly showed multiple intrapulmonary nodules (33/40) and solitary nodules in 7 cases (7/40). Tumor maximum diameters ranged from 3 to 70 mm, with a median of 19 (12, 35) mm. Grossly, all lesions appeared as grayish-white nodules with ill-defined margins and mucoid cut surfaces. Microscopically, tumor cells showed centrifugal distribution around blood vessels, arranged in irregular nests; local mucoid degeneration and chondroid matrix were noted. Intracytoplasmic vacuoles with red blood cells were noted in some tumor cells, indicating primitive vascular lumen differentiation. At the molecular level, WWTR1-CAMTA1 gene fusion was identified in 36 cases and YAP1-TFE3 fusion in 4 cases. Immunohistochemical results showed diffuse positivity for CD31 (38/38), CD34 (36/40), ERG (40/40) and Fli-1 (40/40), and focal positivity for TFE-3 (4/34). Therapeutic responses of 40 patients were assessed using the Response Evaluation Criteria in Solid Tumors criteria: complete response in 4 cases (10.0%), partial response in 5 (12.5%), stable disease in 7 (17.5%), and progressive disease in 24 (60.0%). Conclusions: PEHE is a rare vascular-derived tumor, radiologically characterized by multiple bilateral pulmonary nodules. It has non-specific clinical manifestations; combined use of highly sensitive and specific endothelial markers and genetic testing helps reach the definitive diagnosis. PEHE has an overall indolent course, with long-term survival in some patients. However, multiple lesions, pleural invasion, and distant metastasis may be linked to worse prognoses.

Objective: To score pheochromocytoma and paraganglioma (PPGL) by using the composite pheochromocytoma and paraganglioma grading system (COPPS), to analyze the correlations of COPPS, the pheochromocytoma of the adrenal gland scaled score (PASS), and the grading system for adrenal pheochromocytoma and paraganglioma (GAPP) with tumor metastasis or recurrence and to explore the relationship between gene mutations and metastasis or recurrence in some PPGL. Methods: Clinicopathological data of the 186 paragangliomas diagnosed from January 2012 to December 2022 at Beijing Friendship Hospital, Beijing, China, the Peking University Third Hospital, Beijing, China, and the First Affiliated Hospital of Shihezi University, Shihezi, China were collected and analyzed. Predictive values of the three systems for tumor metastasis or recurrence were evaluated. Immunohistochemistry was performed using the EnVision staining method. Whole-exome sequencing was used to detect gene mutations in 15 tumors. Results: Among the 186 PPGL patients, there were 93 females and 93 males, age 49 (47, 50) years old. Metastasis or recurrence occurred in 60 cases. 34 of the tumors were located in the retroperitoneum. The maximum tumor diameter was >7 cm in 42 cases. A COPPS score ≥3 was observed in 97 cases (52.2%, 97/186), among whom 53 cases (54.6%, 53/97) experienced metastasis or recurrence. The metastasis/recurrence rate in the COPPS score≥3 group was significantly higher than that in the <3 group (χ2=46.469,P<0.001). Tumor location in the retroperitoneum, presence of large nests of cells, pathological mitosis, spindle cells, capsular invasion, and fat infiltration were all associated with a COPPS score ≥3 (χ2=18.370, 51.730, 8.914,18.750, 62.481, 19.354, all P<0.05). The sensitivity of COPPS for predicting metastasis/recurrence was 88.3% (53/60), while the specificity was 65.1% (82/126). Negative expression of SDHB was observed in 50 cases, and negative expression of S-100 protein was observed in 96 cases. DNA extraction failed to produce qualified DNA in 3 cases; among the remaining 12 tumors that were subject to DNA extraction, 739 mutations (in 658 genes) were detected, including 300 germline mutations (in 265 genes) and 439 somatic mutations (in 408 genes). Related pathogenic germline mutation genes occurred on: SDHA, MDH2, MEN1, EGLN1, RET and SDHB. All 12 patients had more than four pathogenic germline mutations. Somatic pathogenic mutation genes included ATRX, KIF1B, EPAS1, HRAS, NF1, and MAML3. Conclusions: A higher COPPS score (≥3 versus <3) is associated with a higher metastasis/recurrence rate. Its sensitivity for predicting tumor metastasis/recurrence is higher than that of GAPP, while its specificity is higher than that of PASS. The combined use of negative SDHB and S-100 protein expression with COPPS could be used to stratify the risk of metastasis/recurrence in PPGL.

Objective: To investigate the clinicopathological and molecular genetic features of micronodular thymoma (MNT) and micronodular thymic carcinoma (MNC) with lymphoid stroma. Methods: Seventeen cases of micronodular neoplasms with lymphoid stroma diagnosed at the Third Affiliated Hospital of Soochow University, Changzhou, China from March 2017 to October 2024 were collected (16 cases of MNT and 1 case of MNC). Their clinicopathological data were reviewed, and their paraffin-embedded sections were stained with immunohistochemistry (IHC). Three cases of MNT and one case of MNC were also examined using next generation sequencing (NGS). Results: Of the 17 patients with MNT or MNC, 7 were male and 10 were female, age 66 (63, 72)years old. Microscopically, tumor cells were scattered in the lymphoid stroma as solid epithelioid nests. The tumor nests were relatively regular in 16 cases, and the tumor cells were mainly short spindle or oval, with bland morphology. Tumor cells of MNC showed marked cytological atypia, conspicuous nucleoli, frequent mitotic figures, and fibrous stroma. IHC showed that tumor cells of all 17 cases diffusely expressed CKpan, CK19 and CK5/6. CD5, CD117 and Glut1 were diffusely positive in the MNC case, but negative in any of the 16 MNT cases. In contrast, there were a considerable number of immature T lymphocytes positive for CD99, TdT and CD1a in the lymphoid stroma around MNT tumor nodules, but not in the MNC. Among the 3 MNT cases subjected to NGS testing, 2 cases had missense mutations in GTF2I (p.L424H), and 1 had missense mutations in HRAS (p.G13V and p.L120P). In the MNC case subjected to NGS testing, gene alterations such as a frameshift mutation in BRCA2 (p.D1451Ifs*12) and a missense mutation in TP53 (p.I195T) were detected, but not GTF2I gene mutations. Conclusions: MNC cells are more atypical than MNT cells. CD5, CD117 and Glut1 are diffusely expressed in MNC, but not in MNT. The genetic alterations in MNC are more diverse than those of MNT, but MNC lacks the characteristic GTF2I mutation of MNT.

Objective: To investigate the prognostic stratification value of MLH1 promoter methylation in a mismatch repair deficiency (dMMR)-type endometrioid endometrial carcinoma (EEC). Methods: A total of 338 patients with confirmed diagnosis of dMMR EEC at Third Hospital of Peking University Health Science Center, from July 2005 to June 2023 were analyzed. Based on the promoter methylation, they were classified into a dMMR methylated (dMMR MET) group (177 cases) and a dMMR nonmethylated (dMMR nonMET) group (127 cases). Somatic mutations were analyzed by targeted sequencing (196/425-gene panel), and transcriptomic differences were assessed by RNA sequencing (Master panel). We compared the clinicopathological characteristics, gene mutation/expression profiles, and molecular pathway activities systematically between the two groups. Results: Compared with the dMMR nonMET group, patients of the dMMR MET group were older significantly [(56.89±8.85) vs. (53.76±9.45) years, P=0.003] and had tumor size of larger diameters [(3.39±1.78) vs. (2.71±1.31) cm, P=0.014]. The menopausal proportion (66.9% vs. 48.8%, P=0.002) and the proportion with tumor buddings (47.5% vs. 30.4%, P=0.036) were higher. No significant differences were identified in FIGO stage, histologic grade, depth of myometrial invasion, lymphovascular invasion, or rate of lymph node metastasis (P>0.05). Mutational profiling revealed that the nonMET group had significantly higher mutation frequencies in CHD4, NF1, SMARCA4, and RET (P<0.05). Transcriptomic analysis demonstrated upregulation of immune-related genes (CCL21, CXCL2) in the MET group, and downregulation of epithelial-mesenchymal transition (EMT)-associated genes (SOX2, FOXA1). Both GO and KEGG enrichment analyses of different gene expression in the MET group demonstrated an association with the MAPK pathway. However, Hallmark pathway analysis showed no significant differences in overall pathway activity between the two groups. Survival analysis revealed no significant differences in progression-free survival (P=0.206) or overall survival (P=0.813) between the groups. Conclusions: The methylation status of the MLH1 promoter has limited value in predicting the prognosis of dMMR EEC. Molecular pathways heterogeneity between the methylated and nonmethylated subgroups suggests the necessity of integrate multi-dimensional indicators to optimize stratification strategies, instead of relying on a single epigenetic marker.

Objective: To investigate the expression of STAT6 and HER2 in triple negative breast cancer (TNBC) and their relationship with clinicopathological features and prognosis. Methods: A total of 189 TNBC samples, including 32 cases with tumor and surrounding normal tissues from Peking Union Medical College Hospital from 2000 to 2011 were collected and tissue microarrays were made. The expression of STAT6 in tumor cells and normal breast tissues was evaluated by immunohistochemistry, and the difference between them were analyzed. The expression of HER2 was classified into 0 and low expression. The relationship with clinicopathological parameters and the prognosis was analyzed. Results: The 57.1% (108/189) cases showed STAT6 high expression, 34.4% (65/189) showed HER2 low expression, and 27.0% (51/189) showed co-expression. The expression of STAT6 in normal breast tissue was lower than that in tumor (t=-5.549, P<0.01). Chi-square test showed that compared with STAT6 low patients, STAT6 high had higher HER2-low, and lower distant metastasis rate (17.3% vs. 47.2%, P<0.01; 22.3% vs. 39.2%, P=0.013); compared with HER2-0 patients, HER2-low had higher STAT6-high, and lower distant metastasis rate (46.0% vs. 78.5%, P<0.01; 16.1% vs. 36.7%, P=0.004). Follow-up was 70(54,95) months, 7 patients (7/189, 3.7%) were lost to follow-up, 68 patients (68/189, 36.0%) relapsed, and 36 patients passed away (36/189, 19.0%). Kaplan-Meier survival analysis showed that tumor size, American Joint Committee on Cancer (AJCC) stage, lymph node metastasis, distant metastasis, and STAT6-low/HER2-0 were negatively correlated with the disease-free survival (DFS) and the overall survival (OS, all P<0.05). STAT6-high, HER2-low expression and STAT6-high/HER2-low were positively correlated with DFS and OS (all P<0.05). Age was only positively associated with OS (P=0.030). COX regression analysis showed that TNBC patients with higher AJCC stage had significantly shorter DFS and OS (HR=8.415, 95%CI: 3.623-19.544, P<0.001; HR=15.377, 95%CI:3.508-67.395,P<0.001); TNBC patients with STAT6-high/HER2-low had significantly longer DFS and OS, which was an independent risk factor for prognosis (HR=0.362, 95%CI: 0.186-0.705, P=0.003; HR=0.168, 95%CI: 0.038-0.743, P=0.019). Conclusions: In triple-negative breast cancer, the expression of STAT6 is higher than that in normal breast tissue. High expression of STAT6 is significantly correlated with HER2-low expression. STAT6-high/HER2-low may predict a good prognosis.

Objective: To investigate FGFR1 mRNA expression and gene characteristics in breast cancer tissue. Methods: Breast cancer samples from 45 female patients (10 HER2-positive, 15 triple-negative and 20 HR-positive/HER2-negative cases) were collected from Peking Union Medical College Hospital between December 2022 and January 2024. FGFR1 mRNA expression was identified using reverse transcription droplet digital PCR, and the FGFR1 gene characteristics was analyzed from 30 samples by fluorescence in situ hybridization. Results: In the group of HER2-positive, 2 cases (2/10) of high FGFR1 mRNA expression and one case (1/7) of high FGFR1 gene amplification were found. Five tumors (5/15) with high FGFR1 mRNA expression, one (1/10) with gene amplification and one (1/10) with low-level amplification of FGFR gene were identified in triple-negative breast cancer. Among HR-positive/HER2-negative samples, high FGFR1 mRNA expressions were confirmed in 4 cases (4/20), with no FGFR1 gene amplification (0/13). Additionally, 3 cases (3/10) showed high FGFR1 mRNA expression in HER2-low expression (IHC 1+and IHC 2+/FISH-) samples. Conclusions: High FGFR1 mRNA expression is observed across different molecular subtypes of breast cancer. High-level FGFR1 gene amplification is uncommonly detected; therefore, further studies with large amount samples are required.

The traditional paradigm of pathology diagnosis faces challenges like data fragmentation and inefficiency in the era of big data and artificial intelligence, necessitating a digital transformation built upon high-quality, standardized databases. This article reviews global efforts in digital pathology database construction and details our team's pilot practice in developing a specialized breast pathology database. We share our approach to case enrollment, systematic data collection, and standardized slide digitization. Key issues, including data schema design, multi-center integration, scanning protocols, and collaborative models, are discussed to inspire industry-wide standardization and support the sustainable development of digital pathology and artificial intelligence in China.

In breast pathology, tumor protein 63 (p63) is a highly sensitive and specific immunohistochemical marker for myoepithelial cells. It plays a crucial role in distinguishing benign from malignant diseases and in differentiating in situ carcinoma from invasive carcinoma. Aberrant p63 expression has also been increasingly recognized as a subject of clinical and diagnostic interest. A comprehensive understanding of the biological characteristics of p63, appropriate selection of antibodies, and its variable expression patterns across different breast diseases is essential for the accurate application and interpretation of p63 immunohistochemistry, enabling the avoidance of diagnostic pitfalls, minimizing misinterpretations, and ultimately supporting more precise pathological diagnosis.

Neuroendocrine tumors are rare, low-grade malignant tumors with a gradually increasing incidence rate in recent years, with gastroenteropancreatic neuroendocrine tumors being the most common among them. The majority of gastropancreatic neuroendocrine tumors have already metastasized to the liver at the time of initial diagnosis; therefore, treatment targeting liver metastases is crucial. The most effective treatment for hepatic metastases of gastropancreatic neuroendocrine tumors is surgical resection. However, 60%-70% of patients are ineligible for radical resection due to diffuse liver involvement. Thus, an alternative treatment option offered for these patients is liver transplantation. Liver transplantation is considered an indication for well-differentiated, unresectable hepatic metastases of gastropancreatic neuroendocrine tumors because of the tumor's low invasiveness, slow growth, and the fact that the liver is often the only distant metastasis site. This article reviews the research progress of liver transplantation therapy for hepatic metastases in gastropancreatic neuroendocrine tumors.

Tumor-associated macrophages (TAMs) are abundant immune cell content in the tumor microenvironment of hepatocellular carcinoma and are associated with tumor progression and therapeutic resistance. This article describes novel TAM subpopulations and potential targets in hepatocellular carcinoma that have been identified based on single-cell and spatial omics technologies. Additionally, it proposes TAMs' classification according to their functions into immunosuppressive, lipid-metabolizing, angiogenic, liver-resident, and immune-stimulating types and summarizes four major therapeutic strategies targeting TAMs, providing a reference for novel TAM-targeted therapies.

Objective: To identify the active ingredients of modified-Zengshengping (ZSP-M) that inhibit the progression of oral squamous cell carcinoma (OSCC) through salivary metabolomics and computer virtual screening, and to screen potential key pharmacodynamic substances. Methods: An experimental tongue OSCC model induced by 4-nitroquinoline-1-oxide (4NQO) was established in 20 C57BL/6 mice. The experiment was divided into a normal group, a 4NQO group, a Zengshengping-original (ZSP-O) group, and a ZSP-M group (n=5). After 24 weeks, the number and volume of tongue tumors were recorded, and the tongue tissue was subjected to HE staining to calculate epithelial structure and cellular characteristic scores. Twelve SD rats were divided into a treatment group and a control group. They were administered ZSP-M or distilled water via gavage, and saliva samples were collected three days later. The prototype components of ZSP-M in saliva were analyzed and identified using ultra-high-performance liquid chromatography-tandem mass spectrometry. The prototype components were further subjected to computer-based absorption, distribution, metabolism, excretion, toxicity prediction, and molecular docking to screen potential key pharmacodynamic substances of interest. The impact of these potential key pharmacodynamic substances on the proliferation of human tongue squamous cell carcinoma cell lines CAL27 and SCC25 was evaluated using cell counting kit-8 assay. Results: The tumor incidence of OSCC in the ZSP-M group of C57BL/6 mice [(1.60±0.24) tumors] was significantly lower than that in the 4NQO group [(2.60±0.24) tumors] (P<0.05), and the pathological score of cytological features [(5.40±0.55) points] was significantly lower than that in the 4NQO group [(7.40±0.55) points] (P<0.05). A total of 16 prototype components entered the saliva, with 10 in positive ion mode and 6 in negative ion mode. Through computer-aided virtual screening, maackiain (MA), scutellarin (SC), chlorogenic acid (CGA), and vitexin-2-O-rhamnoside (VOR) were identified as potential key pharmacodynamic substances with strong binding potential to target proteins. In vitro experimental results showed that all four key pharmacodynamic substances could inhibit the proliferation of CAL27 and SCC25 at 48 and 72 hours (all P<0.05). Compared to the control group, the most significant proliferation inhibition occurred at a high concentration of 100 μmol/L (P<0.01), with the inhibitory effect weakened when the concentration was reduced to 10 μmol/L. Among them, MA demonstrated the most outstanding effect, with an SCC25 cell inhibition rate of (15.16±0.28)% after 72 hours of treatment with 100 μmol/L concentration (P<0.001). Conclusions: ZSP-M can inhibit the development of experimental OSCC, and MA, SC, VOR, and CGA have been identified as potential key pharmacological substances for ZSP-M to enter saliva and exert anticancer activity, reflecting the characteristics of multi-component and multi-target treatment in traditional Chinese medicine formulas.

Ameloblastoma is a locally invasive benign tumor of odontogenic epithelial origin. Research into its molecular mechanisms is hindered by the lack of unified norms for normal control tissue selection, which undermines data comparability. Current studies employ highly heterogeneous control tissues, including tooth germs, pericoronal tissue, normal oral mucosa and even commercial samples, thereby complicating data integration and potentially affecting the accurate interpretation of disease mechanisms. Based on principles of tooth development and the concept of "developmental arrest" in ameloblastoma, we propose a three-tier reference framework for normal control tissue selection: Tier 1 controls (preferred) include normal tooth germs (bell stage prioritized), reduced enamel epithelium and dental lamina remnants among other odontogenic epithelial components, which provide optimal consistency in developmental origin and stage; Tier 2 controls (alternative) consist of site-matched oral mucosa or specific odontogenic cysts linings and may be served as supplements but require explicit limitation disclosure; Tier 3 controls (not recommended), such as tissues of unknown origin or xenogeneic sources, should be avoided due to limited biological comparability. We also propose complementary quality control and normalized reporting recommendations encompassing tissue source verification, histopathological validation, and ethical compliance documentation. This reference framework establishes a reproducible and comparable strategy for controlling tissue selection in ameloblastoma research, thereby contributing to enhanced data reliability and cross-study comparability, while providing methodological reference for other odontogenic tumors and developmental disorders.

Objective: To investigate the expression of miR-199a-5p in intrahepatic cholangiocarcinoma (ICC) and its regulatory mechanism in tumor invasion and metastasis. Methods: Tumor tissues and adjacent normal tissues from 38 ICC patients (aged 40-80 years, including 14 males) were collected at the First Affiliated Hospital of Nanhua University from January 2020 to April 2023. Differential genes and target genes were screened using transcriptome sequencing and multi-database bioinformatics analysis. Transcriptome sequencing was performed to detect the expression of miR-199a-5p and hypoxia-inducible factor 1α (HIF-1α). At the same time, clinical and pathological data such as patients' TNM staging and pathological differentiation were collected to analyze the correlation between the expression levels of miR-199a-5p and HIF-1α and clinicopathological features. Transcriptome sequencing was used to detect the RNA expression of miR-199a-5p and HIF-1α in tumor tissues and adjacent non-cancerous tissues of ICC patients. Immunohistochemistry was employed to detect HIF-1α expression in the tissues. Human normal bile duct epithelial cells, HuCC-T1 and RBE cell lines were cultured and used. The targeting relationship between miR-199a-5p and HIF-1α was verified by dual-luciferase reporter assay. The expression levels of miR-199a-5p and HIF-1α at the cellular level were detected using real-time quantitative PCR (qRT-PCR). RBE cells were divided into three groups through transfection using Lipofectamine 3000 reagent: control group, miR-199a-5p mimic group, and miR-199a-5p inhibitor group. The transfection efficiency and HIF-1α mRNA expression levels were assessed by qRT-PCR. Western blot analysis was used to detect the protein expression levels of HIF-1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 9 (MMP9), and Cleaved caspase3. Cell migration and invasion abilities were assessed using wound healing and Transwell assays. HIF-1α knockdown cell models were established in RBE cells by transfecting them with HIF-1α-specific small interfering RNA (si-HIF-1α), with a negative control group (si-NC) set up simultaneously. After verifying the knockdown efficiency by qRT-PCR and Western blotting, changes in biological behavior of cholangiocarcinoma cells following HIF-1α suppression were observed by examining the expression of proliferation-related protein PCNA, performing wound healing assays, and conducting Transwell migration and invasion assays. This was aimed at clarifying the central role of HIF-1α in cholangiocarcinoma migration and invasion, further supporting that miR-199a-5p exerts its tumor-suppressive effect by targeting and inhibiting HIF-1α. All experimental data were repeated three times. Results: Transcriptome sequencing and multi-database analysis identified miR-199a-5p and HIF-1α as targets. Dual-luciferase reporter assays confirmed that miR-199a-5p directly targets the 3'-UTR of HIF-1α. Transcriptome sequencing tissue expression analysis showed that compared with adjacent non-cancerous tissue, the expression of miR-199a-5p was decreased in ICC (FPKM: 1.24±0.31 vs 3.87±0.56), while the expression of HIF-1α was significantly increased (FPKM: 18.76±6.23 vs 56.15±8.42) (both P<0.05). Immunohistochemistry results showed high expression of HIF-1α in ICC. Analysis of clinical pathological features indicated that ICC patients in the low miR-199a-5p expression group and the high HIF-1α expression group both had later TNM staging and poorer pathological differentiation (both P<0.05). Mechanistically, overexpression of miR-199a-5p downregulated PCNA and MMP9 and upregulated Cleaved caspase 3, while inhibition of miR-199a-5p led to the opposite changes (all P<0.05). Overexpression of miR-199a-5p inhibited the expression of HIF-1α and suppressed cell migration and invasion, whereas inhibition of miR-199a-5p produced the opposite effects (all P<0.05). After knocking off HIF-1α, both the number of migrating and invading cells decreased (both P<0.05). Conclusion: miR-199a-5p is downregulated in ICC and suppresses tumor migration and invasion by directly targeting HIF-1α and regulating downstream molecules involved in proliferation, invasion and apoptosis.