Assessment of miR-760 expression as a potential diagnostic biomarker in oral squamous cell carcinoma: A RT-PCR study

Abstract

Background:

Oral squamous cell carcinoma (OSCC) is an important global health burden and is often diagnosed at an advanced stage. MicroRNAs (miRNAs) are evolving as key regulators of cancer development and progression, with potential roles as diagnostic and prognostic biomarkers. Among them, microRNA-760 (miR-760) has shown tumour-suppressive activity in several cancers; however, its role in oral squamous cell carcinoma remains unclear.

Objective:

The objective of this study was to evaluate the expression pattern of miRNA-760 in tissues of oral squamous cell carcinoma compared to normal oral mucosa using quantitative real-time polymerase chain reaction (qRT-PCR), and to assess its potential clinical utility as a diagnostic biomarker.

Methods:

Thirty histologically confirmed oral squamous cell carcinoma tissue samples and 20 healthy oral mucosal tissue samples were analysed. Total ribonucleic acid was extracted from formalin-fixed paraffin-embedded sections, converted to complementary deoxyribonucleic acid (cDNA), and subjected to (qRT-PCR). Small nuclear RNA U6 was used as the internal reference gene. Delta cycle (ΔCt) threshold values were calculated, and relative expression of miR-760 was determined using the method described as ‘two to the power of negative delta cycle threshold (2^−ΔΔCt)’. The Mann–Whitney U test was used to compare the two groups statistically.

Results:

The expression of miR-760 was significantly higher in oral squamous cell carcinoma tissues. The mean (ΔCt) threshold value for the OSCC group was 1.93 ± 4.04, compared to –1.56 ± 5.11 in the healthy control group (P < 0.001). Relative quantification using the (2^−ΔΔCt) cycle threshold method showed a 1.79-fold up-regulation of miR-760 in OSCC tissues. These findings differ from its downregulation in other malignancies and suggest a possible tissue-specific function in oral carcinogenesis.

Conclusion:

The up-regulation of miR-760 was found in OSCC tissues. This proves that the distinct expression pattern highlights its potential role as a biomarker for early detection. Further studies are needed to explore its biological role and clinical relevance in oral cancer.

INTRODUCTION

Oral squamous cell carcinoma (OSCC) is a major public health concern worldwide, ranking consistently among the six most common malignancies.[1] In South Asia, especially India, OSCC represents nearly one-third of all cancer cases. This high prevalence is primarily linked to modifiable risk factors, such as smoking, tobacco chewing, dietary habits, excessive alcohol intake, and infection with human papillomavirus (HPV).[2,3] Socioeconomic inequality further compounds this burden by delaying access to medical care, contributing to late-stage diagnosis and poorer clinical outcomes.[4]

Although advances in surgery, radiotherapy, and chemotherapy have improved treatment approaches, the prognosis for OSCC remains poor, largely due to late-stage presentation at the time of diagnosis.[5] Early lesions are often asymptomatic and are frequently mistaken for benign oral conditions. These factors, combined with a lack of public awareness about the early signs of oral cancer, result in significant delays in seeking professional care.[6] Continued exposure to high-risk behaviours, such as tobacco and alcohol use, along with the increase in incidence of HPV-associated OSCC, further limits early detection and effective disease management.[3] This scenario highlights an urgent need for dependable, non-invasive biomarkers that can detect OSCC at an early stage, improving prognosis and treatment outcomes.[7]

Recent research suggests that microRNAs (miRNAs) hold great promise as biomarkers in oncology. These small, non-coding RNA molecules are involved in the post-transcriptional regulation of gene expression and are known for their stability in tissues and body fluids, making them ideal candidates for minimally invasive diagnostic tools.[8] Altered miRNA expression can influence multiple cancer-related processes, apoptosis, including cell proliferation, invasion, angiogenesis, and metastasis.[8] In OSCC specifically, dysregulated expression of miRNAs, such as miR-21 and miR-106a, has been well-documented, and these changes are associated with tumour development and progression.[9,10]

Among emerging candidates, miR-760 has gained attention for its tumour-suppressive roles in other malignancies. Studies have reported its downregulation in gastric and colorectal cancers, where reduced expression was linked to poor prognosis and aggressive tumour behaviour.[11,12] miR-760 is thought to regulate pathways, such as GRK-interacting protein-1 transcription and BATF3/AP-1/cyclin D1 signalling, both of which are involved in cell cycle control and apoptosis.[11,12] However, its expression profile and clinical significance in OSCC remain unclear. To date, no comprehensive investigation has addressed this gap, representing an important area for scientific exploration.

Our study aims to evaluate the expression levels of miR-760 in OSCC tissue samples in comparison with healthy oral tissue controls. The goal is to assess whether miR-760 is differentially expressed in OSCC and to explore its potential role as a diagnostic or prognostic biomarker. A better understanding of miR-760 expression in oral cancer may not only contribute to the growing body of literature on miRNA-based biomarkers but also support the development of early detection tools and personalised treatment strategies. Such insights could ultimately enhance clinical outcomes for patients diagnosed with OSCC.

MATERIALS AND METHODS

Study design

This study followed a case–control, cross-sectional design to evaluate the expression of miR-760 in OSCC. The research was conducted at the Department of Dentistry in collaboration with the Central Research Laboratory for Biomedical Research, Vinayaka Mission’s Medical College and Hospital, Karaikal. The primary objective was to compare the expression levels of miR-760 in OSCC tissues and healthy oral tissues, aiming to assess its potential diagnostic value in oral cancer.

Study population and sample collection

The case group included 30 formalin-fixed paraffin-embedded (FFPE) OSCC tissue blocks, comprising 28 samples from buccal mucosa lesions and two samples from tongue lesions. The control group consisted of 20 FFPE tissue samples from clinically healthy buccal mucosa, collected during routine dental procedures unrelated to malignancy. OSCC samples were histopathologically confirmed before inclusion. Control samples were obtained only from individuals without any history or evidence of oral malignancy or precancerous conditions.

Sample size

In the absence of previously published data on miR-760 expression in oral squamous cell carcinoma, a formal a priori sample size calculation could not be undertaken. Nonetheless, the selected cohort comprising 30 OSCC cases and 20 controls was judged to be appropriate. Post hoc statistical analysis revealed that the study achieved approximately 75% power (α =0.05, two-tailed) to detect the observed intergroup difference, supporting the suitability of the sample size for an exploratory biomarker investigation.[13]

Ethical approval

This ethical approval for the study was received from the Institutional Ethical Committee of Vinayaka Mission’s Medical College and Hospital, Karaikal.

RNA extraction

Total RNA was isolated from FFPE tissue sections. Ten 5-µm-thick sections from each block were collected in sterile 2 mL microcentrifuge tubes containing RNA later solution to stabilise the RNA. Samples were incubated at room temperature for 30 minutes, followed by storage at –80°C until extraction. RNA was extracted using the RNeasy FFPE Kit (Qiagen, USA) according to the manufacturer’s protocol. The purity and concentration of RNA were assessed using a NanoDrop spectrophotometer (Nabi, U2 microdigital). Only RNA samples with A260/280 ratios between 1.8 and 2.0 were used for downstream applications.

cDNA synthesis

Complementary DNA (cDNA) was synthesised from the extracted RNA using the Polyadenylation and Reverse Transcription reagent kit (Takara, Japan). All procedures followed the manufacturer’s instructions. Synthesised cDNA was stored at –80°C until further use.

qRT-PCR analysis

Expression of miR-760 was quantified using real-time quantitative polymerase chain reaction (qRT-PCR). Amplification was carried out using the TB Green Advantage qPCR Premix reagent kit (Takara, Japan) on a QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). U6 small nuclear RNA (snRNA) was used as the internal reference gene for normalisation. The following primers were used for amplification

- miR-760

Forward primer: GGCTCTGGGTCTGTGGG

Reverse primer: GAACATGTCTGCGTATCTC

- U6

Forward primer: CTCGCTTCGGCAGCACA

Reverse primer: AACGCTTCACGAATTTGCGT.[14]

The cycling protocol included an initial denaturation at 95°C for 10 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing/extension at 60°C for 20 seconds. A dissociation curve analysis was performed at the end of each run to confirm amplification specificity.

Primer specificity was validated by melting curve analysis, which demonstrated distinct, single peaks for both miR-760 and U6 snRNA, indicating specific amplification without primer–dimer formation. Amplification efficiencies were within acceptable ranges: 97.5–98.2% for miR-760 and 98.7–99.1% for U6 snRNA. The melting temperatures (Tm) for all primers ranged from 58.7°C to 60.5°C, and expected product sizes were 70–85 base pairs, consistent with predicted targets.

Data analysis and normalisation

miR-760 expression levels were quantified using the comparative cycle threshold (Ct) method, also known as the 2^−ΔΔCt method.[15] First, the Ct value of miR-760 in each sample was normalised to that of U6 to calculate the ΔCt (ΔCt = Ct_miR-760 – Ct_U6). Then, ΔΔCt values were calculated by subtracting the mean ΔCt of the control group from the ΔCt of each OSCC sample (ΔΔCt = ΔCt_case – ΔCt_control). Fold change in expression was calculated using the formula 2^−ΔΔCt. Relative expression values were normalised to the control group, which was assigned a baseline value of 1.0.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics (version 27, IBM Corp, USA). The Shapiro–Wilk test was used to assess data normality. As the expression data did not follow a normal distribution, differences between OSCC and control groups were analysed using the Mann–Whitney U test. The Fisher’s exact test was applied for categorical variables, such as gender and anatomical site distribution. AP- value less than 0.05 was considered statistically significant.

RESULTS

Demographic and clinical characteristics

FFPE tissue samples were analysed from 30 patients with oral squamous cell carcinoma (OSCC) and 20 individuals with clinically healthy oral mucosa. The mean age of OSCC patients was 54.16 ± 11.29 years, while that of controls was 48.50 ± 16.14 years, showing no statistically significant difference (P = 0.183). Gender distribution was comparable between groups, with 90% of OSCC patients and 80% of controls being male (P = 0.416). Most OSCC lesions (93.34%) were located on the buccal mucosa, and histopathological analysis revealed 97% were well-differentiated squamous cell carcinoma.

Stability of the reference gene

Cycle-threshold (Ct) values for the internal control U6 small nuclear RNA were tightly clustered and showed no significant inter-group variation (P = 0.798), confirming the suitability of U6 for normalising miR-760 expression [Table 1].

Table 1.

Ct Values for miR-760 and U6 snRNA in OSCC and controls

Group	miR-760 Ct (mean±SD)	U6 Ct (mean±SD)	n
OSCC Cases	28.15±2.85	25.47±4.35	30
Controls	31.48±1.47	31.43±3.71	20

OSCC=Oral squamous cell carcinoma, SD=Standard deviation

Raw amplification signals for miR-760

The mean Ct for miR-760 was significantly lower in OSCC tissue than in normal mucosa (28.2 ± 2.9 vs 31.5 ± 1.5; Mann–Whitney U = 66.0, P < 0.001), indicating greater template abundance in malignant specimens [Table 1].

Normalised expression (ΔCt)

After normalisation to U6, OSCC samples exhibited a markedly lower median ΔCt (1.95) than controls (–1.35). Inter-quartile ranges were non-overlapping, and the group difference remained highly significant (Mann–Whitney U = 55.0, P < 0.001) [Table 2; Figure 1]. Because a lower ΔCt corresponds to higher expression, these data confirm pronounced up-regulation of miR-760 in tumour tissue.

Table 2.

Normalised miR-760 expression (ΔCt) in OSCC and control tissues

Group	n	Mean ΔCt	SD	Median ΔCt	Min	Max	P
OSCC Cases	30	1.93	4.04	1.95	–6.75	12.62	<0.001
Controls	20	–1.56	5.11	–1.35	–6.80	11.87

OSCC=Oral squamous cell carcinoma, SD=Standard deviation

Figure 1.

Box-and-whisker plot of ΔCt values (miR-760 – U6) for OSCC and controls

Relative quantification (2^-ΔΔCt)

Using the comparative Ct approach, the mean inter-group ΔΔCt was + 3.49, corresponding to a 1.79-fold increase in miR-760 expression in OSCC relative to normal mucosa [Table 3].

Table 3.

Relative miR-760 expression (2^-ΔΔCt) in OSCC versus controls

Group	Mean ΔCt	ΔΔCt vs. Control	Mean fold change (2^-ΔΔCt)	Interpretation
OSCC Cases	1.93	+3.49	1.79	Up-regulated
Controls	–1.56	Reference	1.00	Baseline

OSCC=Oral squamous cell carcinoma

The distribution of individual fold-change values is illustrated in Figure 2, which shows a clear right-shift for the tumour group.

Figure 2.

Box-and-whisker plot of fold-change (2^-ΔΔCt) in miR-760 expression

DISCUSSION

OSCC remains a significant threat to human life, particularly in a low-income country like India, where more people use tobacco and betel nut. Despite treatment advances, OSCC survival remains low due to late diagnosis, aggressive tumours, and frequent recurrence. Finding reliable molecular biomarkers is an essential step for the early detection and reducing the risk of OSCC progression in advance. miRNAs are seeking more attention in the current scenario for disease prediction and identification. In our study, we have demonstrated that miR-760 is significantly up-regulated in OSCC tissues compared to normal oral mucosa, providing valuable insights into the potential role of miR-760 in OSCC pathogenesis.

miRNAs have emerged as crucial regulators of various cancer-related processes, including cell proliferation, migration, invasion, and treatment response.[16] MiR-760 has been identified as a tumour suppressor in several cancers, such as gastric,[11] colorectal,[12] breast,[17] lung[18] and hepatocellular carcinomas,[19] where it is typically down-regulated to inhibit cancer progression by repressing cell proliferation and migration. The studies conducted by Ge et al. and Cao et al. in terms of gastric and colorectal cancer have reported that low expression levels of miR-760 were associated with poor prognosis and aggressive tumour features, whereas upregulation of miR-760 would significantly inhibit the tumour cell proliferation and cancer progression.[11,12]

But in OSCC, we found that miR-760 is significantly overexpressed, suggesting that it may function differently in the context of oral cancer. This finding is particularly exciting, as it indicates a tissue-specific role for miR-760, with its potential oncogenic activity in OSCC. Previous research has shown that miRNA functions can vary widely between tissue types depending on the surrounding molecular networks and specific microenvironment.[20]

A study conducted by Liao et al. (2016) demonstrated that miR-760 overexpression promotes proliferation in ovarian cancer by downregulating leucine-rich repeat protein phosphatases (PHLPP2), a key regulator of cell proliferation.[21] This observation further emphasises the divergent roles miR-760 may play in different cancers, acting as an oncogene in ovarian cancer and potentially having a similar role in OSCC. The up-regulation of miR-760 in OSCC could reflect its involvement in oral carcinogenesis by inhibiting tumour-suppressor genes in oral epithelial cells, thereby promoting cellular transformation, migration, and tumour progression. The increased expression of miR-760 may be a compensatory response to oncogenic signalling or altered tumour microenvironmental factors.[22] Similar mechanisms have been observed with other miRNAs, such as miR-21 and miR-155, which function as oncogenic miRNAs in various cancers by targeting tumour-suppressor pathways.[23] Moreover, miR-760 could interact with other non-coding RNAs, including circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), which have been shown to influence miRNA stability and availability.[24] These interactions may help explain the altered expression profiles of miR-760 in OSCC, and further studies on these molecular interactions will provide deeper insights into its regulation in the tumour microenvironment.

The clinical implications of miR-760’s up-regulation in OSCC are significant. miRNAs are stable in body fluids, such as saliva and serum, making them ideal candidates for non-invasive diagnostic biomarkers.[25] Since miR-760 is up-regulated in OSCC tissues, it holds promise as a biomarker for the early detection of oral cancer. Previous studies have demonstrated the diagnostic potential of salivary miRNAs, including miR-21 and miR-125b, in OSCC detection.[10,26,27] If miR-760’s role as an oncogenic miRNA is confirmed, it could serve as a therapeutic target. Antagomirs, synthetic inhibitors of miRNAs, might reverse miR-760’s oncogenic effects, offering a potential strategy for targeted therapy in OSCC.

However, this study has several limitations that warrant further investigation. First, the sample size was relatively small, with only 30 OSCC and 20 control samples. Although sufficient for an initial investigation, larger cohort studies are needed to confirm these findings and explore miR-760’s role across various OSCC stages and grades. Additionally, this study primarily focused on miR-760 expression analysis, without delving into its functional role in OSCC. Future research employing gene knockdown or overexpression models, luciferase assays, and pathway analyses will be essential to identify the direct targets of miR-760 and its downstream molecular effects.

Furthermore, this study has not addressed how miR-760 expression correlates with important clinical outcomes, such as tumour recurrence, metastasis, or survival rates. Correlating miR-760 levels with these clinical parameters would be crucial to determine its prognostic value in OSCC. Additionally, examining miR-760 levels in body fluids, like saliva and serum, could reinforce its potential as a non-invasive diagnostic biomarker and support the development of miRNA-based diagnostic panels for OSCC.

CONCLUSION

This study shows that miR-760 is significantly over-expressed in oral squamous cell carcinoma tissues compared to normal oral mucosa. This finding differs from its known tumour-suppressive role in several other cancers and highlights the tissue-specific nature of miRNA function. The up-regulation of miR-760 in OSCC suggests that it may play an oncogenic role in oral carcinogenesis. Our results also support the potential of miR-760 to be developed as a biomarker for early detection or monitoring of OSCC. However, larger studies and functional investigations are needed to confirm its biological role and clinical value. Overall, miR-760 represents a promising target for future research in oral cancer diagnosis and therapy.

Abbreviations

OSCC—Oral squamous cell carcinoma

miRNA-760—MicroRNA-760

qRT-PCR—Quantitative real-time polymerase chain reaction

FFPE—Formalin fixed paraffin embedded

cDNA—Complementary DNA

PHLPP2—Leucine-rich repeat protein phosphatases

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.