Keywords oral squamous cell carcinoma - microRNAs - gene expression - computational biology
- biomarker
Introduction
Oral squamous cell carcinoma (OSCC) constitutes >90% of all oral cancers and is a
serious health issue globally, as it is highly aggressive. OSCC heavily affects regions
like parts of Southeast Asia and parts of Europe, especially with India accounting
for 45% of all cases around the world.[1 ]
[2 ] The rising incidence of OSCC is largely attributed to lifestyle-related risk factors
such as tobacco use, alcohol consumption, and betel quid chewing. It is now recognized
as the sixth most common cancer among women and the fourth most common in men worldwide.
Despite advances in surgical and radiotherapeutic approaches, OSCC remains difficult
to treat due to its complex molecular landscape, high rates of local invasion, and
a considerable tendency for recurrence. These clinical challenges highlight the urgent
need for molecularly targeted therapies that can improve prognosis and reduce mortality.[3 ]
Among targeted therapies, cetuximab—an estimated glomerular filtration rate inhibitor—was
introduced for OSCC and initially showed promise in combination with chemotherapy
or radiation. However, its overall impact on long-term survival and disease progression
has been limited, and it has not significantly changed the poor prognosis associated
with advanced-stage OSCC. This underscores the need for new, more effective therapeutic
strategies and biomarkers in this disease.[4 ] One of the most commonly altered tumor suppressor genes, TP53 , also known as the “guardian of the genome,” is among the numerous genetic changes
seen in OSCC.[5 ]
[6 ]
[7 ] Uncontrolled cellular proliferation, metastasis, and treatment resistance are all
impacted by the TP53 gene mutations or dysregulation, which interfere with normal cell cycle regulation,
DNA repair processes, and apoptosis. The regulating role of microRNAs (miRNAs) in
the development of cancer has been highlighted by new research.[8 ] Specifically, miR-21 has drawn notice for its carcinogenic potential in several
solid tumors, including OSCC. Through the inhibition of important suppressor genes
like TP53 , increased expression of miR-21 has been associated with increased tumor development,
invasiveness, and resistance to chemotherapy.
To find new biomarkers and explore treatment options that can enhance clinical outcomes
for patients with OSCC, this study intends to examine the molecular interactions between
miR-21 and TP53 gene in OSCC by combining bioinformatics predictions with experimental confirmation.
Materials and Methods
Study Design
This was a cross-sectional observational study aimed at evaluating the regulatory
relationship between miR-21 and TP53 gene in OSCC. A total of 30 OSCC tissue samples and normal tissues were collected
from patients undergoing surgical excision at Saveetha Medical College and Hospitals.
The sample size (n = 30) was determined based on the availability of suitable specimens during the study
period and the exploratory nature of the investigation, in line with similar gene
expression studies.
All samples were confirmed as OSCC through histopathological examination by two independent
oral pathologists. Only newly diagnosed, treatment-naive OSCC cases were included.
Tissues were collected immediately post-resection, snap-frozen in liquid nitrogen,
and stored at −80°C until further use.
Inclusion and Exclusion Criteria
Patients included in the study had histologically confirmed OSCC and provided sufficient,
good-quality tissue samples for molecular analysis. Only treatment-naive individuals
who had not received chemotherapy or radiotherapy were eligible. Exclusion criteria
comprised previously treated or recurrent OSCC cases, inadequate or degraded tissue
samples, and the presence of systemic inflammatory or immunological disorders that
could confound molecular expression profiles.
Identification of a Critical Tumor Suppressor in OSCC and Regulatory miRNAs
To find a tumor suppressor gene known to be relevant in OSCC, a comprehensive study
of the literature was undertaken. Because of its essential function in tumor suppression
mechanisms like apoptosis, DNA repair, and cell cycle regulation, as well as its constant
downregulation in OSCC, TP53 gene was chosen. Potential miRNAs were predicted using two trustworthy online databases,
miRDB and TargetScan, to investigate post-transcriptional control of TP53 gene. Following the selection of miRNAs with a high target prediction score for the
TP53 gene, miR-21 was selected because of its propensity for overexpression in head and
neck cancers and its recognized carcinogenic characteristics.[9 ]
[10 ] RNAfold, an online tool for predicting secondary structure and evaluating the stability
of miRNA hairpin loops, was utilized to further analyze the sequencing and structural
characteristics of miR-21. The stability of miR-21, which is essential for its effective
binding to target mRNAs, was inferred from the minimum free energy (MFE) values produced.
RNA Extraction and Reverse Transcription
Thirty tissue samples were collected from patients with OSCC who had histological
confirmation. TRIzol reagent (Invitrogen, Carlsbad, California, United States) was
used to isolate total RNA from tissue samples in accordance with the manufacturer's
recommended procedure. NanoDrop spectrophotometry from Thermo Fisher Scientific (Massachusetts,
United States) was used to measure the amount and purity of the isolated RNA. RNA
samples were deemed suitable for additional investigation if their 260:280 ratios
fell between 1.8 and 2.0.[11 ] A commercial reverse transcription kit (Takara, Japan) was used to synthesize complementary
DNA (cDNA), which was then kept at −80°C until it was needed for expression investigations.[12 ]
Quantitative Gene Expression Analysis
Using a BioRad CFX96TM real-time PCR machine (California, United States) and SYBR
Green Master Mix (Takara, Japan), quantitative PCR (q-PCR) was used to analyze gene
expression. Eurofins Genomics developed and synthesized specific primers for miR-21
and TP53 gene. β-actin served as a housekeeping gene for TP53 gene normalization, while the short nuclear RNA U6 served as an internal control
for miRNA normalization.
Forty amplification cycles of 95°C for 15 seconds and 60°C for 30 seconds were performed
after initial denaturation at 95°C for 2 minutes in duplicate for each qRT-PCR experiment.
Relative gene expression levels were computed using the 2−ΔΔCq technique, which compares the fold-changes of normal and malignant tissue samples.[12 ]
Outcomes
Primary Outcome
To determine the relative expression levels of miR-21 and TP53 in OSCC tissues using quantitative real-time PCR (qRT-PCR).
Secondary Outcome
To explore the potential inverse regulatory relationship between miR-21 and TP53 based on expression trends and bioinformatics predictions using TargetScan (https://www.targetscan.org/vert_80/ , TargetScan Release 8.0), miRDB (https://mirdb.org/ , version 6.0), and RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ).
Statistical Analysis
To guarantee reproducibility, every experiment was performed in duplicate. The data
were presented using the standard error of the mean (SEM) ± the mean. To compare the
expression levels of OSCC and control tissues, statistical analysis was performed
using GraphPad Prism version 10.1.0 and the Student's t -test. Statistical significance was established at a p -value of less than 0.05.
Ethical Approval
The Institutional Ethics Committee approved this study (Approval No: 581/03/2023/UG/SRB/SMCH,
Department of Medicine, Saveetha Medical College), and all methods followed the Helsinki
Declaration's ethical guidelines. Written informed consent was obtained from all patients
prior to tissue sample collection.
Results
Identification and Structural Analysis of miR-21 as a Regulatory miRNA Targeting Gene
After TP53 gene was discovered to be a major tumor suppressor gene linked to the pathophysiology
of OSCC, we used bioinformatics prediction methods to find regulatory microRNAs that
might affect TP53 gene expression. Several potential miRNAs were found using miRDB and TargetScan.
Because of its previously demonstrated carcinogenic functions in several malignancies,
including OSCC, miR-21 stood out among these as a promising option ([Fig. 1 ]). Previous publications have highlighted the overexpression of miR-21 in head and
neck squamous cell carcinomas, indicating that it regulates genes involved in apoptosis,
cell cycle, and tumor invasion.
Fig. 1 miRNAs predicted to target TP53 gene: Computational prediction of putative miRNAs regulating TP53 gene. miRDB was utilized to generate a comprehensive list of potential miRNAs, highlighting
miR-21 as a significant candidate due to its biological relevance in OSCC and its
expected interaction with TP53 gene.
The secondary structure and thermodynamic stability of miR-21 were predicted using
RNAfold to assess its functional potential in more detail. miR-21 forms a distinctive
and stable stem-loop structure, which is necessary for miRNA processing and function,
according to the computational modelling ([Fig. 2 ]). High thermodynamic stability and a great potential for successful interaction
with target mRNAs like TP53 gene were indicated by the estimated structure's MFE, which was −34.60 kcal/mol ([Table 1 ]). These results lend credence to the theory that miR-21 may contribute to the molecular
mechanisms of OSCC by acting as a strong post-transcriptional regulator of TP53 gene.
Fig. 2 Secondary structure analysis of has-miR-21–3p: RNAfold analysis predicts a stable
stem-loop configuration essential for the proper processing and function of miR-21,
with a measured free energy of −34.60 kcal/mol, indicating strong structural stability.
Table 1
Minimum free energy, mature sequence, match extend, and A + U% of has-miR-21
miRNA
Organism
Minimum free energy
Mature sequence
Match extend
Strand
A + U%
miR-21
Homo
− 34.60 kcal
CAACACCAGUCGA
21/21
3p
50%
Sapiens
UGGGCUGU
Expression Profiling of TP53 Gene and miR-21 in OSCC Tissues
The expression levels of miR-21 and TP53 gene were measured in human OSCC tissue samples (n = 30) and compared with normal tissues (n = 30) using qRT-PCR to validate the in-silico predictions.
According to analysis, TP53 gene expression was considerably lower in OSCC samples than in controls, which is
in line with its well-established function as a tumor suppressor and the fact that
it frequently mutates or is suppressed in cancers ([Fig. 3 ]). In the same OSCC tissues, miR-21 was also significantly elevated ([Fig. 4 ]), supporting its suspected role as an oncogenic miRNA that could aid in TP53 gene suppression.
Fig. 3 Expression profiling of TP53 gene in OSCC tissue samples: Comparative analysis reveals significant downregulation
of TP53 gene expression in patients with OSCC compared with healthy controls, suggesting
its role as a critical tumor suppressor in OSCC pathogenesis.
Fig. 4 Expression levels of miR-21 in OSCC tissue samples: Elevated levels of miR-21 are
observed in patients with OSCC, indicating its potential oncogenic role and interaction
with TP53 gene in the development of oral squamous cell carcinoma.
Further evidence of a regulatory link was provided by the statistically significant
inverse correlation between miR-21 overexpression and TP53 gene downregulation (p < 0.05, Student's t -test). According to this pattern, increased levels of miR-21 may block TP53 gene-mediated tumor suppressive pathways, which could promote tumor genesis, growth,
and treatment resistance.
Expression of miR-21 and TP53 in Relation to Clinicopathological Parameters
To evaluate the clinical relevance of miR-21 and TP53 expression in OSCC, we analyzed their expression profiles in relation to tumor stage,
patient age, tumor grade, and nodal metastasis status using data from TCGA ([Figs. 5 ] and [6 ]) and patient samples ([Figs. 7 ] and [8 ]). miR-21 expression is consistently elevated across all tumor stages, patient ages,
grades, and nodal statuses compared with normal tissues. Although higher median levels
tend to appear in advanced stages and grades, there is considerable variability within
groups, indicating that miR-21 upregulation is a common feature of OSCC independent
of these clinical factors. However, TP53 expression is consistently reduced in HNSCC samples compared with normal tissues
across all clinical categories. This downregulation shows no strong association with
tumor stage, grade, age, or nodal status. In our study, correlation analysis was performed
using Spearman's rank correlation to assess the relationship between gene expression
and clinical parameters. Here, miR-21 expression showed a strong positive correlation with tumor stage, size, and nodal
involvement (p < 0.001). In contrast, TP53 expression was strongly negatively correlated with these clinical parameters (p < 0.001), suggesting inverse regulation with disease progression.
Fig. 5 (A–D ) Expression of miR-21 in HNSCC samples stratified by tumor stage, patient age, tumor
grade, and nodal metastasis status. Box plots show that miR-21 is consistently upregulated
across all clinical subgroups compared with normal tissues, with substantial variability
within each group.
Fig. 6 (A–D ) Expression of TP53 in HNSCC samples stratified by tumor stage, patient age, tumor grade, and nodal metastasis
status. Box plots demonstrate that TP53 is consistently downregulated across all clinical subgroups compared with normal
tissues, with no strong association between expression levels and specific clinicopathological
features.
Fig. 7 Correlation of miR-21 expression with tumor stage, tumor size, and nodal status in OSCC patient samples.
miR-21 expression shows a significant positive correlation with advancing clinical parameters.
Scatter plots illustrate upregulation of miR-21 with disease progression.
Fig. 8 Correlation of TP53 expression with tumor stage, tumor size, and nodal status. TP53 expression exhibits a significant negative correlation, and graphs demonstrate downregulation
of TP53 expression aligned with advancing disease.
Discussion
This study highlights the roles of TP53 gene and miR-21 in OSCC, with around 70% of patients having TP53 gene mutations linked to aggressive tumors.[13 ] Shreya Reddy et al associate high miR-21 levels with advanced cancers, suggesting
it promotes tumor growth by inhibiting TP53 gene.[10 ] Our findings support Rajan et al, emphasizing the need for further research on the
combined effects of multiple miRNAs on tumor behavior.[14 ] This study demonstrates the clinical relevance of miR-21 as a potential biomarker
for early diagnosis and personalized treatment of OSCC. Targeting miR-21 in chemotherapy-resistant
tumors using antagomirs or RNA-based therapeutics may enhance chemosensitivity and
restore TP53 gene activity. Recent advancements in RNA therapeutics show promise for clinical
applications.[15 ] This research enhances understanding of TP53 gene and miR-21 interactions in OSCC, paving the way for novel treatments to improve
patient outcomes.
In addition to these molecular insights, recent advances in immunotherapy, particularly
the introduction of immune checkpoint inhibitors such as nivolumab and pembrolizumab,
have expanded the treatment landscape for OSCC. These therapies have shown promise
in recurrent and metastatic cases by harnessing the immune system to target tumor
cells.[16 ] However, despite these advances, the prognosis for patients with advanced-stage
OSCC remains poor, with limited response rates and many patients not achieving durable
remission. This underscores a considerable unmet need for more effective therapeutic
strategies and predictive biomarkers in OSCC, especially for those diagnosed at later
stages. The identification of molecular regulators such as miR-21 and TP53 may contribute to the development of novel targeted therapies and improve clinical
outcomes in this challenging disease. Although targeted therapies such as cetuximab
have been used in OSCC management, their limited effectiveness in improving survival
outcomes further highlights the necessity for innovative molecular targets, such as
the miR-21/TP53 axis explored in this study.
However, the study has certain limitations. The relatively small sample size may limit
the generalizability of the findings. Additionally, the observational nature of the
study precludes definitive conclusions about causality between miR-21 expression and
TP53 gene downregulation. Functional validation studies, such as gene knockdown or overexpression
experiments, were not performed and are necessary to confirm the mechanistic link.
Moreover, the exclusive focus on miR-21 does not account for the potential influence
of other regulatory miRNAs or signaling pathways involved in OSCC. Future studies
involving larger, multicentric cohorts and functional assays are warranted to validate
and extend these findings.
While TP53 dysfunction is a hallmark of many cancers, including OSCC, its presence or absence
alone may not provide sufficient specificity or sensitivity as a biomarker for early
diagnosis. However, evaluating TP53 expression in conjunction with oncogenic miRNAs such as miR-21 may enhance diagnostic
accuracy and better reflect the molecular mechanisms underlying OSCC progression.
This combinatorial approach could improve the reliability of molecular biomarkers
in clinical practice. The strength of this work lies in proposing an integrated biomarker
strategy that moves beyond reliance on single markers, thereby offering a more robust
and clinically translatable framework for improving early detection and understanding
the molecular complexity of OSCC.
Conclusion
Our study examined the interaction between miR-21 and the tumor suppressor gene TP53 in OSCC. We found that miR-21 is overexpressed, while TP53 gene is downregulated in OSCC tissues, indicating that miR-21 inhibits TP53 gene, which may promote cancer progression. This dysfunction of TP53 gene contributes to uncontrolled growth and metastasis, worsening OSCC aggressiveness.
Our dual approach highlights TP53 's role as a tumor suppressor and miR-21's oncogenic function, emphasizing the need
for further research into miRNA-target interactions as therapeutic targets. Targeting
miR-21 could improve treatment outcomes, and future studies should focus on clinical
applications to enhance diagnostic and therapeutic strategies for OSCC.
