Identification of MicroRNAs Associated with the Diagnosis and Prognosis of Cholangiocarcinoma

ABSTRACT

Introduction:  Cholangiocarcinoma (CHOL) is a malignant neoplasm of the biliary epithelium and represents the second most usual form of liver cancer. Its high aggressiveness and late diagnosis hinder the implementation of effective therapies. The lack of reliable prognostic biomarkers also impairs proper clinical management. In this context, microRNAs (miRNAs) emerge as post-transcriptional regulators of gene expression, with diagnostic and prognostic potential in various tumors.

Objective:  Identify key miRNAs associated with the diagnosis and prognosis of CHOL, with a particular focus on lymph node metastasis, through bioinformatic analysis of data from The Cancer Genome Atlas (TCGA).

Method:  A cross-sectional, descriptive, and exploratory study based on bioinformatics was conducted. A total of 45 samples (36 tumoral and 9 normal) were analyzed from TCGA. The CancerMIRNome, OncomiR, and UALCAN platforms were used for screening, filtering, and differential expression analysis of miRNAs in relation to clinical and demographic variables.

Results:  Two hundred forty-five miRNAs with statistical significance were identified, of which nine were associated with lymph node metastasis: let-7c-5p, miR-1258, miR-182-5p, miR-183-5p, miR-194-3p, miR-301a-3p, miR-378a-3p, miR-92b-3p, and miR-96-5p. Notably, miR-194-3p was associated with all clinical variables analyzed. No significant correlation was found between miRNA expression and overall survival.

Conclusion:  The identified miRNAs demonstrate potential prognostic value in CHOL, particularly for stratifying patients with lymph node metastasis. Further studies involving experimental validation and functional analyses are necessary to confirm their role in CHOL tumor progression.

Key words:
Liver Neoplasms/diagnosis; Cholangiocarcinoma/diagnosis; MicroRNAs; Prognosis; Computational Biology/statistics & numerical data

RESUMO

Introdução:  O colangiocarcinoma (CHOL) é uma neoplasia maligna do epitélio biliar, sendo a segunda principal forma de câncer hepático. Sua alta agressividade e diagnóstico tardio dificultam terapias eficazes. A falta de biomarcadores prognósticos confiáveis também impede o manejo clínico adequado. Nesse contexto, os microRNA (miRNA) surgem como reguladores pós-transcricionais da expressão gênica, com potencial diagnóstico e prognóstico em diversos tumores.

Objetivo:  Identificar os principais miRNA associados ao diagnóstico e prognóstico do CHOL, com enfoque especial na metástase linfonodal, por meio da análise bioinformática de dados obtidos do The Cancer Genome Atlas (TCGA).

Método:  Estudo transversal, descritivo e exploratório baseado em ferramentas de bioinformática. Foram analisadas 45 amostras (36 tumorais e 9 normais) provenientes do TCGA. Para triagem, filtragem e análise da expressão diferencial dos miRNAs em relação às variáveis clínicas e demográficas, utilizaram-se as plataformas CancerMIRNome, OncomiR e UALCAN.

Resultados:  Identificaram-se 245 miRNA com significância estatística, dos quais 9 demonstraram associação com a presença de metástase linfonodal: let-7c-5p, miR-1258, miR-182-5p, miR-183-5p, miR-194-3p, miR-301a-3p, miR-378a-3p, miR-92b-3p e miR-96-5p. O miR-194-3p destacou-se por sua correlação com todas as variáveis clínicas avaliadas. Não foi observada associação estatisticamente significativa entre os miRNA e a sobrevida global dos pacientes.

Conclusão:  Os miRNA identificados apresentam potencial para serem utilizados como biomarcadores prognósticos no CHOL, especialmente na estratificação dos pacientes com metástase linfonodal. Entretanto, são necessários estudos adicionais com validação experimental e análises funcionais para confirmar o papel desses miRNAs na progressão tumoral do CHOL.

Palavras-chave:
Neoplasias Hepáticas/diagnóstico; Colangiocarcinoma/diagnóstico; MicroRNAs; Prognóstico; Biologia Computacional/estatística & dados numéricos

RESUMEN

Introducción:  El colangiocarcinoma (CHOL) es una neoplasia maligna del epitelio biliar y constituye la segunda forma más común de cáncer hepático. Su alta agresividad y diagnóstico tardío dificultan la implementación de terapias eficaces. La falta de biomarcadores pronósticos confiables también impide un manejo clínico adecuado. En este contexto, los microARN (miARN) surgen como reguladores postranscripcionales de la expresión génica, con potencial diagnóstico y pronóstico en diversos tumores.

Objetivo:  Identificar los principales miARNs asociados al diagnóstico y pronóstico del CHOL, con énfasis en la metástasis ganglionar, mediante análisis bioinformático de datos del The Cancer Genome Atlas (TCGA).

Método:  Estudio transversal, descriptivo y exploratorio basado en bioinformática. Se analizaron 45 muestras (36 tumorales y 9 normales) extraídas del TCGA. Se utilizaron las plataformas CancerMIRNome, OncomiR y UALCAN para la selección, filtrado y análisis de la expresión diferencial de miARNs en relación con variables clínicas y demográficas.

Resultados:  Se identificaron 245 miARNs con significación estadística, de los cuales nueve presentaron asociación con metástasis ganglionar: let-7c-5p, miR-1258, miR-182-5p, miR-183-5p, miR-194-3p, miR-301a-3p, miR-378a-3p, miR-92b-3p y miR-96-5p. El miR-194-3p se destacó por su asociación con todas las variables clínicas analizadas. No se observó una correlación significativa con la supervivencia global de los pacientes.

Conclusión:  Los miARNs identificados presentan un valor pronóstico potencial en el CHOL, especialmente en la estratificación por metástasis ganglionar. No obstante, se requieren nuevos estudios con validación experimental y análisis funcionales para confirmar su papel en la progresión tumoral del CHOL.

Palabras clave:
Neoplasias Hepáticas/diagnóstico; Colangiocarcinoma/diagnóstico; MicroARNs; Pronóstico; Biología Computacional/estadística & datos numéricos

INTRODUCTION

Cholangiocarcinoma (CHOL) is a malignant neoplasm originating from the epithelium that covers the biliary ducts and constitutes the second main form of liver cancer, only behind hepatocellular carcinoma¹. Although rare, its incidence has increased over the past decades, especially in the intrahepatic form². This growth is partly attributed to improvements in diagnostic methods and to greater exposure to risk factors, such as primary sclerosing cholangitis, hepatolithiasis, liver parasitic infections (Clonorchis sinensis, Opisthorchis viverrini), and congenital anomalies of the biliary tract²,³. The global incidence of CHOL varies significantly within regions. In Western countries, the age-standardized rate sits within 0.3 and 3.5 cases per 100 thousand people/year, in 1990-2018 series, while endemic Asian regions can reach up to 85 cases per 100 thousand people/year⁴.

Clinically, CHOL is extremely aggressive and usually asymptomatic in the initial stages, which impairs early detection. Most diagnoses occur in advanced stages, when surgical resection with clear margins, the main healing strategy, is no longer viable³. Even with systemic therapies, overall survival remains limited⁵,⁶. In intra-hepatic cholangiocarcinoma (iCCA), the five-year rate is around 9%, reaching up to 40% in cases that can undergo resection⁵. After surgery, however, recurrence rates remained high; in a cohort with 169 patients, the five-year rate was 74.1%, with recurrence-free survival of only 26.1%⁶. The presence of lymph node metastasis is one of the main prognostic factors, consistently associated with lower overall survival and higher risk of recurrence⁷. These data reinforce the need for new diagnostic and prognostic biomarkers to support more effective and individualized strategies.

The clinical CHOL staging, usually based on the American Joint Committee on Cancer modified TNM system, considers the tumoral extension (T), lymph node affliction (N), and the presence of distant metastases (M), classifying patients from stages I to IV⁸. The absence of effective biomarkers for screening, risk stratification, and therapeutic monitoring still represents a central obstacle for managing this disease⁹.

In this scenario, microRNAs (miRNAs) stand out as post-transcriptional regulators of gene expression, modulating processes such as cellular differentiation, apoptosis, metabolism, and oncogenesis¹⁰,¹¹. These small non-codifying RNAs (17–25 nucleotides), when deregulated by genetic or epigenetic mechanisms, may activate tumoral pathways or inhibit suppressor genes, acting as oncomiR or tumoral suppressors¹⁰,¹². Their stability in tissues and fluids, and the specific-tissue expression, confer a high potential as diagnostic and prognostic biomarkers¹³. In the CHOL context, previous studies suggest that miR-21 and miR-25 are differently expressed, the latter being superexpressed in malignant cells and associated with resistance to apoptosis for inhibiting death receptor 4 (DR4)¹⁴,¹⁵. Although these results are promising, further studies are still needed to validate these findings and explore new miRNAs with clinical potential.

The advancement of bioinformatic tools applied to large databases, such as The Cancer Genome Atlas (TCGA), allowed the identification of differently expressed miRNAs in CHOL, favoring their molecular characterization and opening opportunities for precision medicine¹⁶. Despite these advancements, there is still a scarcity of reliable biomarkers for early detection and prognostic stratification of CHOL, limiting effective clinical interventions. In this scenario, the present study aimed at identifying new miRNAs with diagnosis and prognosis potential through integrated analysis of miRNA expression data and clinical information from TCGA-CHOL, with emphasis on the association with lymph node metastasis. In this way, the aim was to contribute to understanding tumor biology and to identify potential targets for future clinical applications.

METHOD

Cross-sectional, descriptive, and exploratory study with a bioinformatic approach, based on publicly available and anonymized data from the TCGA repository¹⁷. A total of 45 biological samples were analyzed, of which 36 came from tumoral tissues of patients with CHOL and 9 from normal control tissues with no evidence of neoplasm, also made available by the TCGA. The molecular data employed in this study corresponded to miRNA (RNA-seq) RNA sequencing, previously normalized as Reads Per Million (RPM), which enables us to compare samples with different sequencing depths. The same set of tumoral and normal data was used in all the platforms employed in this study.

The analytical flow began with a wide screening conducted in CancerMIRNome (version 2.0, accessed in July 2024)¹⁸, a database that integrates TCGA and circulating miRNome data from independent studies, allowing the identification of differently expressed miRNA and the exploration of associations with clinical outcomes and survival. In this step, we assessed differences in expression between tumoral and normal tissues, considering clinical variables such as staging and histological grade, with a significance threshold of p<0.01, which resulted in the identification of 245 differently expressed miRNAs associated with relevant clinical variables.

Next, we used OncomiR (version 1.0, accessed in August 2024)¹⁹, a tool that analyzes miRNA cancer expressions based on TCGA data. The initial screening was conducted through the CancerMIRNome and OncomiR platforms. Through CancerMIRNome, 245 miRNAs with statistical significance (p<0.01) were identified that correlated with clinical variables and survival profile. Then, specific filtering was conducted on OncomiR, incorporating previously assessed and additional clinical variables, like patients’ sex, body mass index, race, and overall survival data. OncomiR was employed in two distinct steps: first, to validate the findings obtained in CancerMIRNome, ensuring consistency of the differential expression; and then, for more detailed stratified analyses of the CHOL subtype. In this phase, statistical tests were applied, including ANOVA and multivariate log-rank, adjusted by False Discovery Rate (FDR), considering significant only the miRNAs with adjusted p<0.01. Only the miRNAs that presented statistically significant differences and overlapped in both platforms were selected, reducing the initial set from 245 to 20 miRNAs with statistical and clinical relevance for the following analyses.

The last step in the analytical flow was conducted on UALCAN (version 2.0, accessed in September 2024)²⁰, a platform that enables stratified analyses of gene and miRNA expressions in TCGA data. The 20 intermediate miRNAs were assessed regarding robustness of differential expression and their association with multiple clinical and prognostic parameters, including sample type (normal tissue versus primary tumor), clinical staging, histological grade, lymph node metastasis status, age, sex, body mass index, and race, in addition to exploratory analysis on overall survival. The relative expression difference was calculated by log2 fold change (log2FC), adopting |log2FC|>1 as a biological relevance criterion, associated with p<0.01. Positive values indicated tumoral superexpression, while negative values reflected greater expression in the normal tissue. After this validation, nine final miRNAs were selected, consistent across platforms and robustly associated with the assessed clinical and prognostic parameters, especially lymph node metastasis.

Finally, an integrative analysis was conducted, in which the selected miRNAs were confronted with previously described data in the literature, aiming to consolidate biological and clinical interpretation, particularly regarding the presence of lymph node metastasis, an important prognostic marker in cholangiocarcinoma.

Considering the investigation was exclusively based on secondary, publicly available, and anonymized data, there was no need for submission to a Research Ethics Committee, in compliance with Resolution N. 510/2016²¹ of the National Health Council and Law N. 12.527/2011²².

RESULTS

Figure 1 presents the sample composition, with the proportion of tumoral and normal tissues (A), as well as the distribution of patients with CHOL following clinical staging (B), including stages I to IV.

Figure 1
Graphic representation of the sample composition analyzed in this study. (A) Proportion between tumoral and normal control samples. (B) Distribution of patients with CHOL following clinical staging

The initial screening identified 245 differently expressed miRNAs among tumoral and normal tissues, selected from the CancerMIRNome and OncomiR platforms. The analyses employed internal tests of differential expression and ANOVA with FDR correction, considering p<0.01 as a threshold and, when applicable, |log2 Fold Change|>1. These miRNAs presented a significant statistical association with relevant clinical variables.

After specific filtering for the CHOL subtype (p<0.01), 20 miRNAs were selected: let-7c-5p, miR-1258, miR-125b-2-3p, miR-129-5p, miR-182-5p, miR-183-5p, miR-194-3p, miR-200b-3p, miR-30c-1-3p, miR-301a-3p, miR-378a-3p, miR-4660, miR-4735, miR-4762, miR-556-3p, miR-6715a, miR-6723, miR-9-3p, miR-92b-3p, and miR-96-5p. To improve the flow of reading, the miRNAs are referred to in the text without the "hsa" prefix.

The differential expression analysis conducted through the UALCAN platform revealed distinct gene regulation profiles. Figure 2 presents a bar graph with miRNAs’ log2 fold change values for tumoral and normal tissues.

Figure 2
Log2 Fold Change bar graph (Tumor vs. Normal) for the analyzed microRNAs (miRNAs). Positive values indicated tumor superexpression, while negative values reflected greater expression in normal tissue. |log2FC|>1 was adopted as a biologically relevant cutoff

Based on the analysis of differential expressions associated with the presence of lymph node metastasis, nine miRNAs with prognostic relevance potential were identified in CHOL. For that end, the platforms CancerMIRNome, OncomiR, and UALCAN were used, with the application of ANOVA with FDR (OncomiR) correction and Student's T test (UALCAN), considering p<0.01 as a threshold. All the selected miRNAs presented statistically significant association with the presence or absence of lymph node metastases (p<0.01); however, no significant correlation was observed between their expression levels and overall patient survival(p<0.05).

Table 1 presents in detail the nine identified miRNAs, accompanied by the respective p values associated with different clinical, demographic, and pathological variables. They all expressed significant differential expression between tumoral and normal tissues (p<0.01) and demonstrated a correlation with relevant prognostic parameters, including clinical staging, histological grade, and lymph node metastasis status. Between the analyzed miRNAs, superexpression was observed in tumoral tissues for miRNAs 182-5p, 183-5p, 301a-3p, 92b-3p, and 96-5p, while let-7c-5p, 1258, 194-3p, and 378a-3p presented higher levels in normal tissues.

Table 1
MiRNA with differential expression associated with nodal metastasis in CHOL

MiRNA	Normal x Tumor	Disease stage	Race	Gender	Age (years)	BMI	Tumoral grade	Nodal metastasis	Survival
let-7c-5p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2, 3, and 4)	p<0.01 (N0, N1)	NS
miR-1258	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-100)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2 and 3)	p<0.01 (N0, N1)	NS
miR-182-5p	p<0.01	p<0.01 (stages 1, 2, and 3)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2 and 3)	p<0.01 (N0, N1)	NS
miR-183-5p	p<0.01	p<0.01	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight)	p<0.01 (stages 2 and 3)	p<0.01 (N0)	NS
miR-194-3p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc., Asian)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2, 3, and 4)	p<0.01 (N0, N1)	NS
miR-301a-3p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-100)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2, 3, and 4)	p<0.01 (N0, N1)	NS
miR-378a-3p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2, 3, and 4)	p<0.01 (N0, N1)	NS
miR-92b-3p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight)	p<0.01 (stages 2 and 3)	p<0.01 (N0, N1)	NS
miR-96-5p	p<0.01	p<0.01 (stages 1, 2, and 4)	p<0.01 (Cauc.)	p<0.01 (M=W)	p<0.01 (41-80)	p<0.01 (normal, overweight, obesity)	p<0.01 (stages 2 and 3)	p<0.01 (N0, N1)	NS

• Captions: BMI = body mass index; NS = non-significant; Cauc. = Caucasian; M = men; W = women, similar statistical significance in both sexes.
• Note: Sample size: Race: Caucasians (n=31); African Americans (n=1); Asians (n=3). Sex: masculine (n=16); feminine (n=19). Age: 21-40 years (n=2); 41-60 years (n=11); 61-80 years (n=20); 81-100 years (n=2).
• Source: Elaborated based on data from the CancerMIRNome, OncomiR, and UALCAN (TCGA) platforms¹⁷-²⁰.

Additionally, the miRNAs’ differential expression was consistent across different population subgroups, encompassing both sexes, 41-100 years age groups, body mass index categories, and, for miR-194-3p specifically, also between individuals from different ethnicities (Caucasians and Asians).

The significant association of miRNAs with multiple histological grades and clinical stages reinforces the hypothesis of their participation in different phases of biliary carcinogenesis, from initial stages to more advanced phases of the disease. Among the assessed biomarkers, miR-194-3p stood out for presenting statistical significance in every clinical and demographic variable analyzed, being a promising candidate to be considered a wide and robust prognostic marker in the context of CHOL.

DISCUSSION

MiRNA differential expression is widely recognized as a central epigenetic mechanism in the regulation of gene expression, influencing essential cellular processes, like proliferation, apoptosis, differentiation, and cellular migration¹⁰-¹². In this context, miRNAs have stood out as crucial epigenetic regulators in several neoplasms, including CHOL²³,²⁴. These molecules act predominantly through post-transcriptional genes, interfering in the translation or stability of the messenger RNA, directly modulating tumoral progression and the biological behavior of neoplasms¹⁰,¹¹.

The findings in the present study reveal a set of nine differently expressed miRNAs with statistical significance, associated mainly with lymph node metastasis, a prognosis marker of high clinical relevance. Among the nine explored, seven (let-7c-5p, miR-182-5p, miR-183-5p, miR-194-3p, miR-378a-3p, miR-92a-3p, and miR-96-5p) had already been correlated with CHOL, and the results of this study are in line with the data previously described in literature²⁵-³⁰. The other two miRNAs addressed in the article (miR-1258 and miR-301a-3p) are new, despite their relationship with other cancer types, mainly hepatocellular carcinoma³¹,³².

In this sense, such miRNAs can be more directly implicated in specific events of local tumoral progression and lymphatic dissemination, exerting limited influence on clinical outcomes in the long term, for instance, mortality. Among them, we highlight let-7c-5p, a member of the let-7 family, known for its oncogenic suppression function²⁵. Previous studies demonstrated that a reduction in the let-7c-5p expression in CHOL tumoral tissues is associated with higher tumoral aggressiveness and inhibition of tumoral self-renewal and growth, although it can also promote invasion and growth in extra-hepatic sites, suggesting a dual role that relies on the cellular context and the molecular targets involved³³. This duality can reflect the interaction with distinct targets, like EZH2 and the DVL3/β-catenin axis, underscoring the complexity of its therapeutic application³³.

MiR-1258 has been characterized as a tumoral suppressor, inhibiting the translation of RTA protein, a regulator of the reactivation of oncogenic herpes viruses, such as the one associated with Kaposi's sarcoma³⁴. In gastrointestinal cancers, reduced levels of miR-1258 have been associated with a higher tumoral stage, lymphovascular commitment, and pathological progression³⁴, which confers prognostic value and therapeutic potential.

MiR-182-5p also stands out as a therapeutic and diagnostic biomarker, associated with mutations in critical genes like BRCA1, BCR-ABL1, and HPGD³⁵,³⁶. Exosomal miRNA mir-182-5p present in the bile of patients with cholangiocarcinoma presented superexpression, promoting tumoral progression by inhibiting the HPGD gene and consequently increasing PGE2, highlighting their potential as a biomarker and therapeutic target²⁶.

MiR-183 regulates crucial cellular cycle pathways, apoptosis, and differentiation³⁷,³⁸. In liver, lung, prostate, and colorectal carcinomas, elevated levels of miR-183 were associated with DNA hypermethylation, greater invasiveness, and worse prognosis³⁷,³⁸, suggesting their utility as a biomarker and therapeutic target.

In this study, miR-194-3p stood out for its association with all the clinical and demographic variables analyzed. Recognized as a tumoral suppressor in several neoplasms, it regulates the epithelial-mesenchymal transition (EMT), the expression of FoxM1, PD-L1/PD-L2, and other pro-tumoral genes³⁹-⁴⁴. Specifically, in CHOL, miR-194-3p inhibits tumoral growth by negatively regulating the transformation sequence of epithelial cells 2 (ECT2) and blocking the Rho pathway, thus configuring as a potential therapeutic target²⁷. The reintroduction of these miRNAs demonstrated the ability to inhibit proliferation, migration, immune evasion, and EMT in different models, indicating their promising potential for therapeutic strategies, including in chemo-resistant tumors³⁹-⁴².

MiR-301a, in turn, acts predominantly as an oncomiR, inhibiting tumoral suppressors like RUNX3, compromising the function of NK cells, and favoring tumoral proliferation⁴⁵-⁴⁷. Despite its contextual effects on the prognosis, this study observed no significant correlation between its expression and patient survival.

MiR-378a-3p demonstrated to be associated with normal suppression, inhibiting RAB31 and the Hedgehog pathway, reducing proliferation, migration, and formation of tumoral stem cells⁴⁸,⁴⁹. Its performance as a diagnostic and prognostic biomarker has been promising in prostate cancer, for instance.

MiR-92b-3p presented an oncomiR profile, being associated with a worse prognosis in hepatocarcinoma, breast cancer, and other neoplasms, modulating targets like CPEB3, ACADL, and Smad7⁵⁰,⁵¹. Similarly, miR-96-5p, a member of the miR-183 cluster, exhibited tumoral superexpression in several types of cancer, repressing suppressor genes, like FOXO3a and PDCD4, and promoting cellular proliferation and survival⁵²-⁵⁷. Specifically, in CHOL, recent studies demonstrated that miR-96 exerts an oncogenic function, favoring tumoral progression and metastasis by inhibiting the MTSS1 gene, suggesting its potential as a prognostic biomarker or therapeutic target, after external validation³⁰.

Collectively, the results reinforce the relevance of an integrative approach that combines bioinformatic analyses and experimental validation to clarify the functional role of these miRNAs. The nine differently expressed miRNAs seem to work in processes like EMT, extracellular matrix remodeling, immune evasion, and proliferation, central mechanisms in the lymphatic dissemination of CHOL. Thus, this study supports the hypothesis that these miRNAs modulate tumoral aggressiveness, presenting potential for prognostic stratification of patients with CHOL. Although they were not correlated with overall survival, they demonstrated a strong association with lymph node metastasis, one of the main determinants for prognosis and eligibility for surgical resection. Thus, even with no direct impact on mortality, these miRNAs can be more directly implicated in specific events of local tumoral progression and lymphatic dissemination, helping in post-surgical stratification and therapeutic decision-making.

Among the study's limitations, we highlight the absence of experimental validation in vitro or in vivo, restricting the immediate translation of the findings to clinical practice. This is thus an exclusively bioinformatic analysis, with an exploratory character and no external validation. On the other hand, the transdemographic stability suggests that the analyzed miRNAs present biological and clinical robustness, being little influenced by individual characteristics, which reinforces their potential as prognostic markers. Furthermore, the data derived exclusively from TCGA are subject to sample bias and gaps in detailed clinical information. Further investigations shall contemplate the manipulation of miRNA, like miR-194-3p and miR-1258, in cellular and murine models, as well as the validation in independent cohorts, to confirm their applicability as non-invasive biomarkers or therapeutic targets.

CONCLUSION

This study identified nine differently expressed miRNAs associated with the presence of lymph node metastasis in CHOL, highlighting miR-194-3p for its broad correlation with clinical and demographic variables. Although no significant associations with overall survival have been observed, the identified miRNAs demonstrate a potential diagnostic and prognostic value, especially for tumoral stratification and understanding the underlying molecular mechanisms of disease local progression. Additional studies, with experimental validation and functional analysis, are needed to consolidate their utility as biomarkers and potential therapeutic targets to manage CHOL, considering the exploratory character of the present analysis.

• FUNDING SOURCES
  None.

DATA AVAILABILITY STATEMENT

All the contents associated with the article are included in the manuscript.

ACKNOWLEDGMENTS

To Dr. Ruan César Aparecido Pimenta for his valuable lecture on "Introduction to Bioinformatics for Biomedical Research Using Online Platforms", ministered at the Faculdade Atenas Passos’ Oncology Academic League (Liga Acadêmica de Oncologia da Faculdade Atenas Passos), which contributed to delving into the knowledge applied in this study on miRNA in the diagnosis and prognosis of cholangiocarcinoma.

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Edited by

• Associate editor:
  Tatiana de Almeida Simão. Orcid iD: https://orcid.org/0000-0001-8509-2247

• Scientific-editor:
  Anke Bergmann. Orcid iD: https://orcid.org/0000-0002-1972-8777

Publication Dates

• Publication in this collection
  26 Jan 2026
• Date of issue
  2026

History

• Received
  23 July 2025
• Accepted
  05 Oct 2025