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23 July 2026

18 Pages

IGF2-H19 Locus Expression Profile and Biomarker Potential in Oral Cancer

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1
Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia
2
Clinic for Otorhinolaryngology and Maxillofacial Surgery, University Clinical Center of Serbia, 11000 Belgrade, Serbia
3
School of Dental Medicine, University of Belgrade, 11000 Belgrade, Serbia
4
Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
Biology2026, 15(15), 1221;https://doi.org/10.3390/biology15151221
This article belongs to the Special Issue Head and Neck Cancer: Current Advances and Future Perspectives
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Simple Summary

Oral cancer is one of the most common head and neck malignancies, and its early and sensitive detection remains a major clinical challenge. In this study, we investigated several molecules involved in the control of normal cell growth and development to determine whether they could help distinguish cancer tissue from healthy tissue and potentially help in determining prognosis of patients. We analyzed their expression in oral cancer cells grown in the laboratory and in tissue samples obtained from patients with oral cancer. We found that the levels of these molecules were significantly different between non-cancerous and cancer tissues. When all three molecules were analyzed together, they showed improved ability to identify oral cancer. However, these molecules were not associated with predicting disease outcome. Our findings suggest that these biological markers may be useful for improving the detection of oral cancer and could contribute to the development of more accurate diagnostic tools in the future. Further studies involving larger numbers of patients are needed to confirm these results.

Abstract

IGF2-H19 locus is important for normal development and growth, and its deregulation has been implicated in various cancers, with conflicting data in oral cancer. IGF2 and H19 expression was analyzed in oral cancer (SCC-25, SCC-15) and normal keratinocytes (HaCaT) cell lines, as well as in cancer tissue and adjacent non-cancerous tissues from 55 patients with oral cancer. The expression of IGF2 and H19 was increased in oral cancer cell lines compared with control cell line. In clinical samples, expression of the IGF2-H19 locus and its associated hsa-miR-675-5p was significantly lower in cancer tissue compared with adjacent non-cancerous tissue. IGF2, H19, and hsa-miR-675-5p demonstrated moderate ability to discriminate between oral cancer and non-cancerous tissues, while the combined analysis of all three molecules improved diagnostic performance. Our results suggest that IGF2-H19 and hsa-miR-675-5p may have potential as molecular biomarkers for discriminating between oral cancer and non-cancerous tissue, but do not appear to have prognostic value.

1. Introduction

Head and neck squamous cell carcinoma (HNSC) is a heterogenous group of cancers arising in the head and neck region, including oral cavity cancer [1]. Oral cancer (oral squamous cell carcinoma, OSCC) is the most common subtype within this group, accounting for approximately 90% of all HNSC cases [2]. The global incidence of oral cancer is increasing [3], particularly in Europe and among younger populations [4]. Lifestyle factors such as smoking and alcohol consumption, as well as environmental factors like infection with high-risk human papillomavirus types, are considered important triggers for malignant transformation of the squamous cells in oral cavity [2]. Oral cancer is usually diagnosed at an advanced stage of the disease, which significantly worsens the prognosis [5]. Surgical treatment is the primary therapy often followed by radiotherapy with or without chemotherapy [6]. However, local recurrences occur in up to 30% of cases indicating the need for more sensitive and specific methods to distinguish oral cancer from normal oral mucosa. This indicates the importance of integrating molecular pathology approach alongside conventional histopathological analysis. Furthermore, the identification of prognostic biomarkers as well as discovery of novel therapeutic targets is essential to improve oral cancer patient outcomes and enable more personalized treatment in the future.
IGF2-H19 locus (11p15.5) represents an imprinted locus encoding growth factor IGF2 and long noncoding RNA (lncRNA) H19, in which paternal IGF2 and maternal H19 expression are tightly regulated, while dysregulation of this locus has been implicated in the pathogenesis of multiple human cancers [7]. Proper tissue growth during embryogenesis is enabled due to balanced expression of IGF2 and H19. Loss of imprinting leads to overexpression of IGF2, deregulation of IGF2-H19 and consequently to malignancy. IGF2 expression was increased in adrenocortical carcinoma [8], hepatoblastoma [9], and other cancer types, thus considered as protooncogene. The available evidence on IGF2 in OSCC remains limited. Previous studies conducted on two commercial cell lines, SCC-4 and SCC-25, both derived from tongue epithelium, have reported increased IGF2 expression. However, these studies did not investigate the biological significance of IGF2 overexpression or clarify its potential role in OSCC development and progression [10]. H19 is also mainly recognized as a protooncogene, but there are data suggesting its tumor suppressor role depending on the cellular context, indicating a dual role for H19 [11]. There are conflicting data on H19 expression in oral cancer [12,13]. A recent systematic review confirmed that H19 is dysregulated in OSCC. However, the available studies report conflicting results, with some demonstrating increased and others decreased H19 expression. Therefore, H19 may not be suitable as a standalone biomarker in OSCC [14].
Micro RNAs (miRNAs) are encoded within IGF2-H19 locus. MiRNAs are small non-coding RNA molecules, ~24nt in length, with an important role in the regulation of vital cellular processes, such as differentiation and apoptosis. miRNAs regulate expression of target genes by suppressing expression posttranscriptionally either by mRNA degradation or translational repression. It is known that hsa-miR-483 and hsa-miR-675 miRNAs are transcribed from the IGF2-H19 locus. Hsa-miR-483-3p is located within the second intron of the IGF2 gene, while hsa-miR-675-5p is embedded within the first exon of H19 [7]. Aberrant expression of these miRNAs has been detected in different cancer types [8,15,16,17,18,19].
To the best of our knowledge, IGF2-H19 locus and its mRNA, lncRNA and miRNAs have not been extensively studied in oral cancer. Considering the role of IGF2-H19, we hypothesized that molecules transcribed from the IGF2-H19 locus contribute to oral cancer development cooperatively, and thus mRNA, lncRNA and miRNAs generated from the locus might be promising molecular candidates for sensitive discrimination of cancer from non-cancerous tissue as well as prognosis in oral cancer patients. This study aimed to comprehensively analyze expression levels of IGF2 and H19 and their associated miRNAs in oral cancer cell lines, publicly available datasets and oral cancer clinical samples and to evaluate their association with clinicopathological characteristics and biomarker potential. Thus, this study fills a gap in knowledge regarding IGF2-H19 locus-related transcripts and their biomarker potential in oral cancer.
The main finding of the current study is that the combination of IGF2, H19, and hsa-miR-675-5p shows good performance in distinguishing between oral cancer and non-cancerous tissue.

2. Materials and Methods

2.1. Cell Cultures

In this study, three cell cultures were utilized, including the human oral squamous cell carcinoma (OSCC) cell lines SCC-25 and SCC-15 obtained from the American Type Culture Collection (ATCC CRL-1628™, ATCC CRL-1623™) as well as immortalized human keratinocytes HaCaT (CLS/Cytion, catalog number 300493, Eppelheim, Germany). HaCaT cells represented a healthy keratinocyte cell line and were used as the control group in this study.
Cells were cultured in T25 cell culture flasks using a complete medium consisting of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 11320-033) supplemented with 10% fetal bovine serum (FBS; American Type Culture Collection, Manassas, VA, USA), 100 U/mL penicillin–streptomycin solution (Antibiotic–Antimycotic solution (100×; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 400 ng/mL hydrocortisone (Thermo Fisher Scientific, Waltham, MA, USA) for OSCC cell lines. All cell cultures were maintained under standard conditions in a humidified atmosphere containing 5% CO2 at 37 °C. The complete medium was replaced every second day. Upon reaching 70–80% confluence, cells were passaged.

2.2. Public Database Analysis

Data on expression of IGF2, H19, and their associated hsa-miR-483-3p, and hsa-miR-675-5p were obtained from The Cancer Genome Atlas (TCGA) HNSC cohort via Xena platform (https://xenabrowser.net/, accessed on 12 February 2026) [20]. Expression data in oral cancer and normal tissue were generated by RNASeq using the Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA). Dataset version 2017-10-13 was used for IGF2 and H19, whereas dataset version 2017-09-08 was used for hsa-miR-483-3p, and hsa-miR-675-5p. Samples were filtered by anatomic neoplasm subdivision, and larynx, hypopharynx, oropharynx and tonsil were filtered out. The final dataset consisted of 347 (87.2%) primary tumor samples and 51 (12.8%) normal solid tissue samples. Data were downloaded for further statistical analysis. The total number of analyzed samples varied between candidates because expression data were missing for some samples.

2.3. Study Group and Biological Material

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Faculty of Medicine of the University of Belgrade (approval number 1550/VII-6, 18 July 2019) and the Ethics Committee of the University Clinical Center of Serbia (approval number 1880/71, 25 December 2025). From all included patients, written informed consent was obtained. The study group consisted of 55 patients diagnosed with oral cancer. All patients underwent surgical treatment at the Clinic for Otorhinolaryngology and Maxillofacial Surgery, University Clinical Center of Serbia, between 2018 and 2024. All patients received adjuvant radiotherapy, while some also underwent adjuvant chemotherapy, depending on clinical indications. Demographic and clinical information of the study group is presented in Table 1. The median follow-up time was 17 months. Overall survival was defined as the time from oral cancer diagnosis to death from any cause and was presented in months. Data on overall survival time were unavailable for two patients who died and were therefore excluded from the survival analysis. Patients who were alive at the last follow-up were censored at that time.
Table 1. Characteristics of the study group.
During surgery, both tumor tissue and adjacent non-cancerous tissue (located at least 2 cm from the tumor edges) were collected. Non-cancerous tissue was histologically confirmed as non-malignant by the pathologist. Tissue samples were immediately preserved in RNAlater (Invitrogen, Waltham, MA, USA) and stored at −80 °C.

2.4. RNA Isolation

From cultured cell lines SCC-25, SCC-15 and HaCaT, total RNA was isolated by using TRIzol reagent (Invitrogen, Waltham, MA, USA) following manufacturer’s protocol. Total RNA was isolated from clinical samples by mirVana™ miRNA Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA). Isolated RNA was stored at −80 °C. Concentration and purity of total RNA was measured at nanodrop (Implen, Munich, Germany). Relative expression of mRNAs and mature form of miRNAs was performed by qRT-PCR using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA).

2.5. Reverse Transcription and Quantitative Real Time PCR (RT-qPCR)

For relative expression analysis of IGF2 and H19 gene, cDNA was synthesized by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) following the recommended protocol by the manufacturer. The reaction mix consisted of 10× RT buffer, 10× RT random primers, 25× dNTP mix, MultiScribe Reverse Transcriptase and 20ng of RNA in the final volume of 15 µL. To relatively quantify expression of miRNAs of our interest, cDNK was synthesized by using the TaqManTM microRNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and pool of stem-loop primers following the manufacturer’s instructions. The thermal cycling conditions for cDNA synthesis were as follows: 30 min at 16 °C, 30 min at 42 °C, 5 min at 85 °C followed by a hold at 4 °C. All synthesized cDNAs were stored at −20 °C until qPCR.
Relative quantification of the expression levels of IGF2, H19, their hosted miRNAs, hsa-miR-483-3p and hsa-miR-675-5p, and endogenous controls GAPDH and RNU6B was performed using TaqManTM gene expression assays or TaqManTM microRNA gene expression assays (Thermo Fisher Scientific, Waltham, MA, USA): Hs00277496_s1 (IGF2), Hs00262142_g1 (H19), Hs99999905_m1 (GAPDH), ID 002339 (hsa-miR-483-3p), ID 002005 (hsa-miR-675-5p), ID 001093 (RNU6B). No AmpErase UNG Universal PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) was used in the qPCR reaction mix. Temperature profile was as follow: 5 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All reactions were run in triplicate.
Relative expression of the mRNA, lncRNA and miRNAs were determined using the dCt method (ΔCt = Ctsample − Ctendogenous control), and reported as 2−ΔCt. Results were presented as mean ± standard deviation (SD).
Experimental validation of both miRNAs in both cell lines and clinical samples was not feasible because of technical and resource constraints. Specifically, hsa-miR-483-3p was measured only in cell lines, whereas hsa-miR-675-5p was analyzed only in clinical samples. Results from the relative quantification of hsa-miR-675-5p expression have been previously published for a subgroup of 35 oral cancer patients [21], while in the current study, expression was evaluated in an enlarged group of patients (n = 48).

2.6. Statistical Analysis

Obtained data were analyzed using SPSS v22.00 (IBM SPSS Statistics, Armonk, NY, USA) and GraphPadPrism v9.0 software (GraphPadPrism software, Boston, MA, USA). Normality of data distribution was tested by Kolmogorov–Smirnov test and Shapiro–Wilk test. For normally distributed data, comparisons of relative expression were performed using the Student’s t-test. Data that showed not normal distribution were analyzed by non-parametric statistical Mann–Whitney test. Differences in relative expression of mRNAs and miRNAs between oral cancer and paired non-cancerous tissue were analyzed by Wilcoxon sign-rank test. Bonferroni correction was applied for multiple testing. Correlation between relative expression levels of IGF2, H19 and their hosted miRNAs was assessed using Pearson’s correlation test for normally distributed data and Spearman’s correlation test for non-normally distributed data. Associations between expression and clinical characteristics were determined by the χ2 test. Discriminatory potential was estimated by Receiver operating curve (ROC) with Area under the curve (AUC) and 95% confidence interval (95% CI). Optimal cut-off value was determined by maximal Youden index, and for cut-off value specificity and sensitivity were calculated. Positive predictive value (PPV) and negative predictive value (NPV) were calculated based on the sensitivity and specificity values obtained at the optimal cut-off. Given the design of the current study with an equal number of cancer and non-cancerous samples (1:1 ratio), the calculated PPV and NPV reflect the diagnostic performance within the study cohort and do not represent population-based predictive values based on disease prevalence. Predicted probability from the logistic regression model based on combination of IGF2 and H19 to distinguish between oral cancer and non-cancerous tissue was calculated as follows: logit(p) = 0.143 + (−0.259 × 2−ΔCt IGF2) + (−0.064 × 2−ΔCt H19); combination of IGF2, H19 and hsa-miR-675-5p was calculated as follows: logit(p) = 0.540 + (−0.308 × 2−ΔCt IGF2) + (−0.060 × 2−ΔCt H19) + (−1.255 × 2−ΔCt hsa-miR-675-5p). Oral cancer patients were dichotomized according to the expression of IGF2, H19 and hsa-miR-675-5p in cancer tissue into low and high expressed group based on median of expression. Overall survival between patients with low and high expression was compared by log-rank test of the Kaplan–Meier survival curves. Hazard ratio for outcome was calculated by Cox proportional regression analysis with 95% CI. Power of the study was calculated by G*Power 3.1 software [22]. p values were two-tailed and if less than 0.05 were considered significant.

3. Results

3.1. Expression Analysis of IGF2, H19 and hsa-miR-483-3p in Oral Cancer Cell Lines

Expression levels of IGF2, H19 and hsa-miR-483-3p were evaluated in oral cancer cell lines SCC-25 and SCC-15 (Figure 1). Compared with the control HaCaT cell line, both IGF2 and H19 showed significantly elevated expression levels in the OSCC cell lines (p = 0.0003, p < 0.0003, respectively, Mann–Whitney test, Bonferroni correction). Similarly, hsa-miR-483-3p expression was significantly upregulated in the oral cancer cell lines compared to the control (p = 0.0003, Mann–Whitney test, Bonferroni correction).
Figure 1. Expression profile of IGF2 (a), H19 (b) and hsa-miR-483-3p (c) in oral cancer (OSCC: SCC-25, SCC-15) and control cell line (HaCaT). Representative micrographs of healthy keratinocytes and OSCC cell lines, acquired using an inverted light microscope at 10× magnification, are shown in panel (d). Data are presented as mean ± standard deviation (SD). Statistical significance is shown with symbols: *** p < 0.001, **** p < 0.0001.

3.2. Public Database Analysis of IGF2, H19, hsa-miR-483-3p and hsa-miR-675-5p Expression and Its Biomarker Potential

To evaluate expression of IGF2, H19, hsa-miR-483-3p and hsa-miR-675-5p in oral cancer and normal tissue, we performed analysis of the public TCGA-HNSC dataset (Figure 2). IGF2 was slightly elevated in tumor but without significance compared to normal tissue (p = 1.000, t-test, Welch’s and Bonferroni correction). There was no significant difference in expression level of H19 and hsa-miR-675-5p in tumor compared to normal tissue (p = 0.588; p = 1.000, respectively, t-test, Welch’s and Bonferroni correction). Level of hsa-miR-483-3p was significantly upregulated in cancer tissue (p = 0.0008, t-test, Welch’s and Bonferroni correction).
Figure 2. Expression profile of IGF2 (a), H19 (b), hsa-miR-483-3p (c) and hsa-miR-675-5p (d) in oral cancer and non-cancerous tissue, retrieved from the TCGA-HNSC dataset. Data are presented as mean ± standard deviation (SD). Statistical significance is shown with symbol: *** p < 0.001.
When paired samples from the TCGA-HNSC dataset were analyzed, a consistent expression trend was observed for IGF2, H19, hsa-miR-483-3p and hsa-miR-675-5p (Supplementary Figure S1). However, a statistically significant difference between cancer and paired non-cancerous tissues was detected only for hsa-miR-483-3p, which was significantly upregulated in cancer tissue compared with non-cancerous tissue (p = 0.008, t-test, Bonferroni correction).
In the same dataset, significant but moderate correlation in IGF2 and H19 expression in cancer tissue was observed (Pearson’s rho = 0.204, p = 0.0001), while strong correlation between H19 and hsa-miR-675-5p was observed (Spearman’s rank rho = 0.653, p < 0.0001). There was also significant correlation of IGF2 and hsa-miR-483-3p expression in cancer tissue (Spearman’s rank rho = 0.512, p < 0.0001), Supplementary Figure S2.
Potential of IGF2, H19, hsa-miR-483-3p and hsa-miR-675-5p to discriminate between oral cancer and non-cancerous tissue was estimated by ROC analysis (Figure 3). Among the analyzed candidates, only hsa-miR-483-3p demonstrated moderate discriminatory potential as a biomarker (AUC = 0.692, 95% CI = 0.595–0.788, p = 0.0005), whereas no significant discriminatory performance was observed for IGF2, H19 and hsa-miR-675-5p.
Figure 3. IGF2 (a), H19 (b), hsa-miR-483-3p (c) and hsa-miR-675-5p (d) potential to discriminate between oral cancer and non-cancerous tissue. ROC analysis was performed on data retrieved from the TCGA-HNSC dataset. The red line indicates reference.
None of the analyzed IGF2, hsa-miR-675-5p and hsa-miR-483-3p were associated with overall survival according to Kaplan–Meier analysis and therefore do not appear to have prognostic value (Supplementary Figure S3). However, there was a significant difference in overall survival between patients with low and high H19 expression in cancer tissue (p = 0.010, log-rank test). Patients with low H19 expression had worse overall survival.

3.3. Expression Analysis of IGF2, H19 and hsa-miR-675-5p in Oral Cancer Clinical Samples and Association with Clinicopathological Characteristics

The expression profiles of IGF2, H19, and hsa-miR-675-5p in clinical samples from oral cancer patients are presented in Figure 4 and Supplementary Figure S4. A significant difference in relative expression between oral cancer tissue and adjacent non-cancerous tissues was observed for IGF2 (p = 0.021, Wilcoxon signed-rank test, Bonferroni correction) and H19 (p = 0.039, Wilcoxon signed-rank test, Bonferroni correction). Both IGF2 and H19 showed significantly decreased expression in oral cancer tissues compared to non-cancerous tissues, exhibiting approximately 3.5-fold and 3.9-fold lower expression levels, respectively. Similarly, hsa-miR-675-5p—which is transcribed from H19—was also downregulated in oral cancer compared to adjacent non-cancerous tissue (p < 0.0003, Wilcoxon signed-rank test, Bonferroni correction), showing 3.4-fold decrease in expression. Based on 55 paired oral cancer and non-cancerous tissue samples, the post hoc calculated power of the study was 0.99, assuming an effect size of 0.8 and a significance level of alpha 0.05.
Figure 4. Expression profile of IGF2 (a), H19 (b) and hsa-miR-675-5p (c) in oral cancer and adjacent non-cancerous tissue. Data are presented as mean ± standard deviation (SD). The y-axis is on log10 scale. Statistical significance is shown with symbols: * p < 0.05, *** p < 0.001.
Strong positive correlation was noticed between IGF2 and H19 expression in oral cancer tissue (Spearman’s rho = 0.601, p < 0.0001). In group of 48 clinical samples, there was weak, non-significant negative correlation between H19 and hsa-miR-675-5p expression in oral cancer tissue (Spearman’s rho= −0.013, p = 0.929), Supplementary Figure S5.
Association of IGF2, H19 and hsa-miR-675-5p expression in tumor tissue with demographic and clinicopathological characteristics of oral cancer patients is presented in Table 2. Recurrences were associated with H19 expression (p = 0.022, χ2 test). No other significant associations were observed with clinicopathological characteristics.
Table 2. Association of IGF2-H19 and hsa-miR-675-5p expression with demographic and clinicopathological characteristics of oral cancer patients.

3.4. Biomarker Potential of IGF2, H19 and hsa-miR-675-5p in Oral Cancer

Figure 5 presents ROC curves demonstrating the discriminatory performance of the analyzed candidates. Both IGF2 and H19 showed moderate, yet promising ability to discriminate between oral cancer and non-cancerous tissue according to results of ROC analysis (AUC = 0.620, 95% CI = 0.516–0.724, p = 0.030, PPV = 56.5%, NPV = 61%; AUC = 0.622, 95% CI = 0.517–0.727, p = 0.027, PPV = 67.5%, NPV = 60%, respectively). The relative expression of hsa-miR-675-5p also showed moderate discriminatory potential for distinguishing oral cancer from non-cancerous tissue (AUC = 0.664, 95% CI = 0.554–0.773, p = 0.005, PPV = 72%, NPV = 62.2%). The combined analysis of IGF2 and H19 improved discriminatory performance compared to individual biomarkers (AUC = 0.644, 95% CI = 0.541–0.747, p = 0.009, PPV = 62.7%, NPV = 64.7%). Importantly, the integration of all three analyzed molecules further enhanced the ability to discriminate between oral cancer and non-cancerous tissue, yielding the highest test performance (AUC = 0.717, 95% CI = 0.616–0.819, p = 0.0002, PPV = 67.3%, NPV = 70.4%). PPV and NPV values reflect test performance within the study cohort.
Figure 5. (a) IGF2 (b) H19 (c) hsa-miR-675-5p (d) combined IGF2 and H19 (e) combined IGF2, H19 and hsa-miR-675-5p potential to discriminate between oral cancer and adjacent non-cancerous tissue. The red line indicates reference.
To evaluate the prognostic potential of IGF2, H19, and hsa-miR-675-5p, Kaplan–Meier survival analysis was performed, and survival curves for low- and high-expression of analyzed candidates were compared (Figure 6). No significant differences in overall survival were observed between patients with low and high expression levels of IGF2, H19, or hsa-miR-675-5p in oral cancer tissue.
Figure 6. Kaplan–Meier curves of overall survival in oral cancer patients depending on IGF2 (a), H19 (b), and hsa-miR-675-5p (c) expression levels. Low and high levels of relative expression refer to values above or below the median. Survival data were missing for two patients; therefore, they were excluded from the survival analysis.
IGF2, H19 and hsa-miR-675-5p cannot be used as independent predictors of mortality in oral cancer patients based on the results of the univariate and multivariate Cox proportional hazard ratio analysis (Table 3).
Table 3. Impact of IGF2, H19 and hsa-miR-675-5p expression on overall survival outcomes in oral cancer patients.

4. Discussion

The purpose of this study was to perform a comprehensive analysis of the relative expression of IGF2, H19, and their hosted miRNAs, hsa-miR-483-3p and hsa-miR-675-5p, originating from the IGF2-H19 locus, in oral cancer cell lines, publicly available datasets and clinical samples. The use of three independent study models indicates the strength of the present study. Cell lines provided a controlled experimental system that enabled the evaluation of gene expression in a homogeneous tumor cell population without the influence of the tumor microenvironment. Publicly available datasets allowed expression evaluation in a larger cohort of patients, while our clinical samples reflected the biological complexity and heterogeneity of OSCC in vivo. The differences observed among these models should not necessarily be considered contradictory; rather, they highlight the complexity of IGF2-H19 regulation and the influence of different biological contexts. Together, these complementary approaches provide a more comprehensive understanding of gene expression patterns than any single model alone.
We first evaluated the expression of IGF2, H19, and hsa-miR-483-3p in commercially available OSCC cell lines and healthy keratinocytes. All three molecules tested in cell lines were significantly upregulated in OSCC compared with control cells, suggesting coordinated deregulation of transcripts originating from the IGF2-H19 locus in oral cancer. Based on our findings in OSCC cell lines, both IGF2, H19 and hsa-miR-483-3p may be considered to have protooncogenic roles. These results are consistent with previous studies reporting the increased level of IGF2 [10] and H19 in oral cancer cell lines [23,24]. It has been demonstrated that H19 promotes metastasis and invasion in tongue cancer cells through the let-7/HMGA2 axis [23]. Furthermore, another study suggested that the increased expression of H19 in oral cancer cell lines is associated with hypomethylation of the H19 promoter [13] indicating the importance of consideration of methylation level.
To further validate and expand these findings, we analyzed publicly available datasets containing gene expression profiles from healthy and tumor oral tissue samples.
Contrary to our observations in cell lines, a statistically significant difference between cancer and non-cancerous tissues was observed only for hsa-miR-483-3p, whereas IGF2 and H19 expression levels did not differ significantly in the TCGA-HNSC cohort. These unexpected findings indicated that the expression patterns observed in established cell lines may not fully reflect those present in clinical specimens. Although hsa-miR-483 is located within the IGF2 genomic locus and generates two mature transcripts—miR-483-5p and miR-483-3p—evidence suggests that its transcription is not solely dependent on IGF2 expression [25]. Consequently, we expanded our investigation by including hsa-miR-675-5p, another microRNA transcribed from the IGF2-H19 locus. Similar to IGF2 and H19, TCGA-HNSC dataset analysis did not reveal statistically significant differences in hsa-miR-675-5p expression between healthy and tumor tissues. Given these inconclusive results and the observed discrepancies between in vitro cell line models and publicly available tissue datasets, we decided to evaluate the expression of IGF2, H19, and hsa-miR-675-5p in our own cohort of clinical OSCC specimens in order to better define their potential involvement in oral carcinogenesis and their value as diagnostic and prognostic biomarkers.
In our study group, IGF2 was significantly decreased as H19 and hsa-miR-675-5p in oral cancer clinical samples. The differences in IGF2 and H19 expression profiles observed between oral cancer cell lines and clinical samples may be attributed to differences in cellular composition, tumor heterogeneity and microenvironment. Cell lines consist exclusively of tumor cells and lack stromal and infiltrating cell populations, whereas expression analysis in clinical samples reflects bulk tissue expression. Therefore, cell lines may not accurately represent the expression profiles observed in clinical specimens. Furthermore, there were differences in IGF2 expression between TCGA-HNSC dataset analysis and our clinical samples. Although the expression trends for H19 and hsa-miR-675-5p were in line with our findings in clinical samples, the TCGA-HNSC dataset analysis did not reveal statistically significant differences in expression between oral cancer and normal tissue samples. This inconsistency may be attributed to differences in methodology, including the use of RT-qPCR in our study and RNA sequencing in the TCGA-HNSC dataset, as well as differences in sample size. Additionally, our study utilized adjacent non-cancerous tissue as a control, which may have influenced the results due to the potential presence of a field of cancerization [26,27,28].
Previous research found no differences in IGF2 expression between normal and cancer tissues in patients with head and neck squamous cell carcinoma, including tumors located in the larynx, pharynx, or oral cavity [29]. The discrepancy between the previous report and our findings may reflect differences in study group, sample size, tumor localization, disease stage, and experimental methodology, all of which could influence IGF2 expression patterns.
Numerous reports showed deregulation of H19 in OSCC. However, data are contradictory, as H19 was identified as both downregulated and upregulated in oral cancer, as reported in recent systematic review [30]. H19 level was significantly decreased in oral cancer tissue compared to non-cancerous tissue in our study which was in line with earlier reported findings [12,31,32]. On the contrary, H19 was increased in tongue cancer tissue compared to adjacent normal tissue, confirming its oncogenic role [23,24]. H19 and hsa-miR-675-5p were reported as upregulated in oral cancer tissue compared to normal tissue from unrelated healthy individuals [30]. In clinical samples of head and neck squamous cell carcinoma—including cancers of the larynx, oropharynx, and hypopharynx—H19 and hsa-miR-675 were reported to be upregulated in tumor tissue compared with noncancerous tissue, and this finding was associated with prognostic significance [33]. These findings contrast to our results, which were limited to oral cavity tumor sites. This discrepancy underscores the importance of considering anatomical sites-specific differences and highlights the heterogeneity of cancers arising within the head and neck region.
H19 and its associated hsa-miR-675-5p show context-dependent expression profile and oncogenic or tumor suppressor function across different cancer types. For instance, H19 expression was increased in tumor tissue of patients with locally advanced rectal carcinoma [34]. Opposite findings for different tumor types, even though all are developed of the epithelial cells, can be explained by context-dependent specificity, as it was shown that H19 behaves as protooncogene, as well as tumor suppressor gene in different tumor tissues. Furthermore, different methylation patterns, loss of imprinting, tumor environment, but also technical and methodological factors might contribute to disparities among different cancer types.
While studies performed on cell lines generally report increased expression of these genes, analyses of tissue samples have produced inconsistent findings, resulting in conflicting data in the literature and raising questions regarding the causes of these discrepancies [32,35]. The results obtained from cell lines reflect strictly controlled experimental conditions and the absence of the tumor microenvironment. Furthermore, OSCC cell lines represent well-established in vitro models derived from aggressive oral squamous cell carcinomas and are capable of long-term propagation under controlled culture conditions. Therefore, their molecular profile may not fully correspond to that observed in clinical specimens. Future studies may benefit from incorporating data obtained from primary cell cultures, which could provide an intermediate model between established cell lines and patient-derived tissues.
One of the aims of our study was to evaluate translational potential of IGF2, H19, and hsa-miR-675-5p expression as biomarkers for the discrimination of cancer and non-cancerous tissues. We have shown that the combination of IGF2, H19, and hsa-miR-675-5p yields the highest test performance for distinguishing oral cancer from non-cancerous tissue. The superior performance of the combined model supports the concept that simultaneous assessment of multiple components of the IGF2-H19 locus may provide complementary diagnostic information and better reflect locus dysregulation than individual biomarkers. The IGF2, H19 and hsa-miR-675-5p biomarker profile could serve as a promising valuable tool to conventional histopathology, potentially aiding in the more precise definition of surgical margins. Also, these findings might potentially contribute to the development of sensitive diagnostic tools necessary to overcome the challenges of late-stage oral cancer detection. Nevertheless, these findings require validation in larger independent cohorts before any conclusions regarding clinical utility can be drawn.
None of the analyzed candidates was considered a potential prognostic biomarker. Results from previous studies on this topic are also conflicting. One previous study identified H19 as significant prognostic biomarker for both overall survival and relapse [34]. The absence of a significant association with overall survival should be interpreted with caution, as the relatively limited sample size and restriction of the cohort to oral cavity tumors may have reduced the power to detect prognostic effects. Additionally, the low number of death events observed during follow-up may have reduced the statistical power of the survival analyses and hindered the detection of potentially relevant prognostic associations. Therefore, the negative findings reported herein should not be regarded as conclusive evidence that these biomarkers lack prognostic relevance. The discrepancy between the TCGA-HNSC and clinical cohort findings regarding the prognostic significance of H19 remains unclear; however, differences in clinicopathological characteristics, treatment strategies, follow-up duration, and analytical methodologies between the cohorts, together with the limited number of death events observed in our study, may have contributed to the divergent findings.
When interpreting results from the current study, it is important to acknowledge several limitations. The limitation of this study is the use of only two OSCC cell lines—SCC-15 and SCC-25, which may not fully capture the biological heterogeneity of oral squamous cell carcinoma. In addition, HaCaT cells were used as the control group; however, as immortalized epidermal keratinocytes, they do not represent a true equivalent of normal oral keratinocytes. Therefore, the results obtained from cell line experiments should be interpreted with these limitations in mind. Furthermore, we used only one endogenous control—GAPDH for mRNA and lncRNA and RNU6B for miRNAs. Therefore, this limitation should be considered when interpreting the diagnostic and prognostic potential of H19, IGF2, and hsa-miR-675-5p. hsa-miR-483-3p and hsa-miR-675-5p were not experimentally validated in both cell lines and clinical samples due to technical and resource constraints. The incomplete validation of both miRNAs across all experimental settings limits our ability to draw firm conclusions regarding the coordinated regulation of the IGF2-H19 locus and should therefore be considered when interpreting the findings. Nevertheless, integration of experimental findings with a publicly available dataset provided additional support for the observed expression profile of IGF2-H19 locus. Although we had a relatively small number of clinical samples, we analyzed data from the TCGA-HNSC project using the Xena platform. Our patient group was limited in size, but it remains a valuable resource, as the number of paired samples was greater compared to the TCGA dataset. Despite the relatively limited sample size, our study group was sufficient to detect differences in IGF2, H19, and hsa-miR-675-5p expression between cancerous and non-cancerous tissues, assuming an effect size of 0.8, statistical power of 0.8, and an alpha level of 0.05.

5. Conclusions

The results of the present study suggest that genes within the IGF2-H19 locus, together with the associated hsa-miR-675-5p expression, may serve as potential molecular biomarkers for sensitive discrimination of oral cancer from non-cancerous tissue. Diagnostic performance was further improved when the expression levels of IGF2, H19, and hsa-miR-675-5p were analyzed in combination. However, validation in larger, independent patient cohorts is required to confirm these findings and to further elucidate the role of IGF2-H19 locus gene expression in oral cancer prior to potential clinical implication. In addition, mechanistic studies using advanced experimental models, including 3D culture systems, are warranted to investigate the biological functions of these genes and the epigenetic regulation of the IGF2-H19 locus in oral cancer.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology15151221/s1, Figure S1: Expression profile of IGF2 (a), H19 (b), hsa-miR-483-3p (c) and hsa-miR-675-5p (d) in oral cancer and non-cancerous tissue, retrieved from the TCGA-HNSC dataset. Data are presented as mean ± standard deviation (SD). Statistical significance is shown with symbol: ** p < 0.01; Figure S2: Correlation analysis between expression levels of IGF2 and H19 (a) IGF2 and its hosted hsa-miR-483-3p (b), and H19 and its hosted hsa-miR-675-5p (c). Correlation analysis was done for expression data in oral cancer tissue. Data were retrieved from the TCGA-HNSC dataset.; Figure S3: Kaplan–Meier curves of overall survival in oral cancer patients depending on IGF2 (a), H19 (b), hsa-miR-483-3p (c), and hsa-miR-675-5p (d) expression levels. All data were retrieved from the TCGA-HNSC dataset. Low and high levels of relative expression refer to values above or below the median; Figure S4: Expression profile of IGF2 (a), H19 (b) and hsa-miR-675-5p (c) in oral cancer and adjacent non-cancerous tissue—paired samples plot. The y-axis is on log10 scale. Statistical significance is shown with symbols: * p < 0.05, ** p < 0.01, *** p < 0.001; Figure S5: Correlation analysis between expression levels of IGF2 and H19 (a), and H19 and its hosted hsa-miR-675-5p (b) in oral cancer clinical samples from our study group.

Author Contributions

Conceptualization, K.Z. and M.J.K.; methodology, K.Z., M.J.K. and M.S.V.; software, K.Z. and J.R.S.; validation, K.Z., M.J.K., M.S.V. and J.R.S.; formal analysis, K.Z. and M.J.K.; investigation, K.Z., M.J.K., M.S.V. and J.R.S.; resources, G.S. and N.T.; data curation, K.Z. and M.J.K.; writing—original draft preparation, K.Z. and M.J.K.; writing—review and editing, M.J.K., G.S., M.S.V., N.T. and J.R.S.; visualization, K.Z. and M.J.K.; supervision, K.Z. and G.S.; project administration, K.Z. and G.S.; funding acquisition, K.Z. and G.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Ministry of Science, Technological development and Innovations of the Republic of Serbia [grant agreement numbers: 451-03-34/2026-03/200178].

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Faculty of Medicine of the University of Belgrade (approval number 1550/VII-6, 18 July 2019) and the Ethics Committee of the University Clinical Center of Serbia (approval number 1880/71, 25 December 2025).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

The graphical abstract was created using Canva Business, utilizing only non-AI-generated design elements. The concept and design were developed entirely by the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
95% CI95% confidence interval
AUCArea under the curve
DMEM/F12Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12
FBSfetal bovine serum
HNSCHead and neck squamous cell carcinoma
HR Hazard ratio
lncRNAlong noncoding RNA
miRNAsmicroRNAs
mRNAMessenger RNA
NPVNegative predictive value
OSCCOral squamous cell carcinoma
PPVPositive predictive value
ROCReceiver operating curve
SDStandard deviation
TCGA-HNSCThe Cancer Genome Atlas—Head and Neck Squamous Cancer
TNMTumor, node, metastasis

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