Exploration of the association between HIF3α mRNA and lncRNA MALAT1 in laryngeal squamous cell carcinoma by correlation analysis
- Authors:
- Published online on: May 1, 2024 https://doi.org/10.3892/ol.2024.14425
- Article Number: 292
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Copyright: © Kyurkchiyan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Laryngeal carcinoma ranks as the second most prevalent type of head and neck cancer, following lip and oral cavity cancer. The Global Cancer Observatory reported 184,615 registered cases of laryngeal malignancies in 2020, with men exhibiting incidence and mortality rates of 3.6 and 1.9 per 100,000 (age-standardized rate), respectively, excluding non-melanoma skin cancer (1). Laryngeal squamous cell carcinoma (LSCC) constitutes the majority of laryngeal tumors, with tobacco smoking, heavy alcohol consumption and human papillomavirus infections identified as primary risk factors for its development; however, some authors dispute the latter in laryngeal cancer (2,3). Despite advances in cancer treatments and the evolution of personalized medicine in oncology, LSCC continues to pose a global health burden, with minimal improvements in survival rates over recent decades (4). Often diagnosed at advanced stages, LSCC significantly impacts the quality of life of affected individuals. Exploration of the molecular biology and genetics of LSCC remains crucial for improving current treatment protocols, which encompass surgery, radiotherapy, chemotherapy and/or EGFR-targeted therapy.
Active cell proliferation, a hallmark of cancer, enhances tumor growth and induces hypoxia due to reduced oxygen supply. Hypoxia, in turn, triggers changes in the expression levels of hypoxia-inducible factor (HIF)α subunits, leading to the formation of HIF transcriptional factors when they bind to HIFβ subunits (5). These HIFs modulate the expression of genes associated with cell proliferation, angiogenesis, migration, invasion, cellular metabolism and cell survival (6). While HIF1α and HIF2α have been extensively studied and linked to the development of malignancies, the detailed investigation of HIF3α, particularly in the context of laryngeal cancer, remains limited (7). Dynamic changes in the levels of the three HIFα subunits in response to varying durations of hypoxia have been observed, with HIF1α activation occurring during acute hypoxia, followed by HIF2α, and subsequent expression of HIF3α during chronic hypoxia, a phenomenon known as the ‘HIF switch’. This switch is purportedly regulated by non-coding RNAs (ncRNAs) (8,9).
The complexity of HIF3α is evident as >10 transcript variants have been identified (10). The presence of these variants adds a layer of intricacy to the function of HIF3α, with HIF3α1 and HIF3α9 bearing the closest sequence identity to HIF1α and HIF2α sequences. Moreover, tissue-specific expression and potential divergent roles in gene expression regulation have been suggested for HIF3α variant transcripts (10,11).
Long ncRNAs (lncRNAs) form a substantial category of RNA transcripts, >200 nucleotides long, and are recognized as potential molecular markers. lncRNAs serve as major regulators of cellular pathways, and have been implicated in cancer development and progression (12). Notably, the lncRNAs MALAT1 and HOTAIR have been extensively studied. Under hypoxic conditions in lung cancer, elevated levels of lncRNA HOTAIR have been associated with direct interaction with HIF1α, leading to increased proliferation, migration and invasion (13). Similarly, the lncRNA HOTAIR and HIF1α axis has been strongly linked to radioresistance in cervical cancer (14,15). Furthermore, lncRNA MALAT1, induced by hypoxia and regulated by HIF1α and HIF2α, has been shown to function as a sponge for microRNA (miR)-3064-5p in breast cancer, promoting cell proliferation, tumor growth and metastasis (16).
We have previously published results on the expression levels of the three HIFα subunits, and both lncRNAs MALAT1 and HOTAIR in LSCC (17,18). Building on this foundation, the present study aimed to explore the connection and relation of lncRNAs with major HIF genes in laryngeal carcinoma. In addition, the study aimed to investigate the association of HIF1α, HIF2α and HIF3α, with the clinicopathological features of patients with LSCC.
Materials and methods
Sample collection
The present study assessed samples from 63 patients diagnosed with LSCC. Tumor and adjacent normal tissue specimens within 3 cm from the border of the tumor were collected during surgical procedures conducted between the periods January 2015-June 2019 and November 2020-August 2023 at the Ear, Nose and Throat Department of UMHAT ‘Tsaritsa Yoanna-ISUL’ (Sofia, Bulgaria). Following surgical excision, the specimens were promptly frozen in liquid nitrogen (−196°C) and transported to the Molecular Medicine Center Biobank (Sofia, Bulgaria) within 1–2 days. The tissue samples were subsequently stored at −80°C until use or for longer durations. Written informed consent was obtained from each patient, which is kept at our repository at the Molecular Medicine Center and the Department of ENT, Medical University (Sofia, Bulgaria). The study was approved by the Ethics Committee of the Medical University of Sofia (approval nos. BK-297/14.04.2015; BK-373 11.04.2016; 7836/17.11.2020 and 4584/04.07.2023). None of the included patients had undergone radiotherapy or chemotherapy before surgery.
Histopathological and clinical characteristics
Additionally, from the same 63 patients with LSCC, formalin-fixed, paraffin-embedded tissue was utilized for to determine the histopathological characteristics of the samples. To fix the tissues 10% neutral buffered formalin for 24–48 h at room temperature was used. The section thickness used was 5–6 µm. The analysis included the gold standard for diagnosis of solid tumors. The histology results reported the type and origin of tissue samples, determination of cancer staging (how advanced is the cancer) and cancer grading (determining the aggressiveness of the cancer). Moreover, the presence or absence of lymph node metastasis and cell differentiation were examined histopathologically. The location of all malignant tumors (anatomical sites infiltrated by the tumor) were observed and determined during surgery. Along with histopathological characteristics, clinical features about sex, age, tobacco smoking, alcohol consumption, familial history and harmful environment were taken.
Total RNA extraction
Total RNA was isolated from fresh frozen normal and tumor tissue samples from each patient using the RNeasy Mini Kit (Qiagen GmbH), according to the manufacturer's protocol. The quality of the RNA was assessed by denaturing electrophoresis on a formaldehyde gel, while the concentration of RNA was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.).
Reverse transcription-quantitative PCR (RT-qPCR)
For cDNA synthesis, 1 µg total RNA from each sample was used, employing the High-Capacity cDNA RT Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the RT2 First Strand Kit (Qiagen GmbH) in accordance with the respective manufacturer's recommendations. High-Capacity cDNA Kit provides improved detection of mRNAs and RT2 First Strand Kit is designed for lncRNA detection. The cDNA samples were subsequently processed for further analysis or stored at −20°C until required. In the present study, the expression levels of HIF1α mRNA, HIF2α mRNA, HIF3α mRNA, were analyzed using 1X RotorGene SYBR Green PCR Mix (Qiagen GmbH), whereas lncRNA MALAT1 and lncRNA HOTAIR were analyzed using RT2 SYBR Green Mastermix (Qiagen GmbH). Due to nature of lncRNAs the manufacturers suggested specific kits for the different aims. A laboratory optimization and analysis were performed of all included in the RT-qPCR techniques to validate the specificity of the reagents. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 15 sec, primer annealing at 55°C for 30 sec and synthesis with data acquisition at 72°C for 30 sec. The 1X QuantiTect Primer Assays (Qiagen GmbH) were used for mRNA detection: Hs_HIF1A_1_SG (NM_001243084), Hs_EPAS1_1_SG (NM_001430), Hs_HIF3A_1_SG (NM_152795), Hs_VEGFA_1_SG (NM_001025366), whereas the RT2 lncRNA qPCR assays for human MALAT1 (NR_002819) and human HOTAIR (NR_003716) (Qiagen GmbH) were used for lncRNA detection. Each sample underwent triplicate examination and corresponding no-template controls were assessed. β-actin (Hs_ACTB_1_SG, NM_001101; QuantiTect Primer Assay; Qiagen GmbH) was employed as the reference gene for normalization. The relative expression of each gene in the tumor samples was determined using the 2−ΔΔCq method, with the calculation of relative quantification (RQ) (19). An RQ ≥2 was defined as upregulation, RQ <0.5 as downregulation, and RQ values between 1.99 and 0.5 indicated no change in expression.
Statistical analysis
IBM SPSS Statistics v.23 (IBM, Corp.) was used for all statistical analyses. The expression levels of the studied targets were evaluated in 63 laryngeal tumor and adjacent normal laryngeal squamous cell tissue specimens. Normality was assessed using the Kolmogorov-Smirnov test, after which appropriate non-parametric tests, including Mann-Whitney U test, Kruskal-Wallis test and Spearman's rank correlation analysis were employed. Additionally, in non-parametric tests, ranks were calculated and were compared in statistical analysis. Additionally, Pearson's correlation analysis and coefficient of determination were utilized for the analysis of linear dependence between variables. P<0.05 was considered to indicate a statistically significant difference.
Results
Study cohort
The present study included 63 patients, comprising 2 women and 61 men, all of whom were diagnosed with LSCC. The mean age of the patient group was 61 years (age range, 41–84 years). The clinicopathological characteristics of the patients and the expression levels of the explored targets are summarized in Table I.
Relative expression in laryngeal tumor samples
Significant dysregulation of the three HIFαs (HIF1α, HIF2α and HIF3α mRNA) and both lncRNAs (MALAT1 and HOTAIR) was observed in LSCC samples compared with in adjacent normal laryngeal specimens. Specifically, HIF1α mRNA was found to be upregulated in 44 out of 63 patients (69.84%) and normally expressed in the remaining 19 patients (30.16%). Additionally, upregulation of HIF2α mRNA was detected in 7 patients (11.11%), while the expression was almost silenced in 24 patients, with the remaining 32 patients exhibiting normal expression levels. Notably, HIF3α mRNA was predominantly downregulated in the majority of patients (48 patients, 76.19%), while 10 patients (15.87%) exhibited normal levels of expression and 5 patients (7.94%) demonstrated significant upregulation. Furthermore, lncRNA MALAT1 showed downregulation in 35 patients (55.56%) and upregulation in 4 patients (6.35%), whereas it was found to have normal expression in the remaining 24 patients with LSCC (38.09%). On the other hand, lncRNA HOTAIR exhibited upregulation in 35 patients with LSCC (55.56%), downregulation in 16 patients (25.40%), and normal expression in 12 patients (19.05%). Comprehensive details of the relative expression levels of the studied targets are published separately in previous original articles by our team (12,13).
Association between HIFα expression levels and clinicopathological characteristics
The associations between the RQ data of the studied HIF1/2/3α mRNAs and the clinicopathological characteristics of patients with LSCC are presented in Table I, along with the results of non-parametric tests (Mann-Whitney U and Kruskal-Wallis tests). Statistically significant associations were demonstrated between HIF3α mRNA expression levels, age and family history, and between HIF1α mRNA levels and tumor localization. Notably, patients aged >60 years exhibited significantly higher HIF3α mRNA expression levels when compared with those ≤60 years (P=0.032). Moreover, higher HIF3α mRNA expression levels were observed in patients reporting a family history of cancer (P=0.047). Additionally, HIF1α mRNA expression levels demonstrated gradual elevation from supraglottis to subglottis tumor location (P=0.010).
Correlation analysis between the expression levels of HIFα mRNA and the lncRNAs MALAT1 and HOTAIR
A correlation analysis was conducted to investigate the strength and degree of dependency among the included targets in samples from the 63 patients with LSCC. Spearman's rank correlation analysis was applied, revealing statistically significant weak and moderate monotonic dependencies between various targets. Positive correlations were observed between the lncRNAs MALAT1 and HOTAIR (rs=0.353; P<0.004); HIF2α mRNA and lncRNA MALAT1 (rs=0.431; P<0.01); HIF3α mRNA and lncRNA MALAT1 (rs=0.347; P=0.005); HIF3α mRNA and lncRNA HOTAIR (rs=0.279; P=0.027); and HIF3α mRNA and HIF2α mRNA (rs=0.285; P<0.025). The only significantly negative correlation was detected between HIF3α mRNA and HIF1α mRNA (rs=−0.341; P=0.006). To further assess any linear relationship between the targets, Pearson's correlation analysis was performed, revealing a moderately strong positive linear dependence between HIF3α mRNA and lncRNA MALAT1 (r=0.630; P<0.001) and a weak linear dependence between both lncRNAs, MALAT1 and HOTAIR (r=0.267; P=0.034). The results indicated that 39.6% of the HIF3α and MALAT1 variables could be predicted to be linearly dependent, with the rest of the coefficient of determinations (R2) at 7.2% or lower. Fig. 1 displays a small network representing the significant dependence between the studied targets, illustrating positive and negative correlations. Additionally, scatter plots with fitted regression lines and R2 values are presented in Fig. 2 to visually represent the relationships between significantly correlated targets.
Discussion
The results of the present study indicated marked dysregulation in the expression levels of HIF1/2/3α mRNAs, and the lncRNAs MALAT1 and HOTAIR, in the studied group of patient samples, suggesting their potential involvement in laryngeal carcinogenesis. The primary focus of the present study was to assess the correlations between the three investigated HIFα mRNAs and the hypoxia-related lncRNAs MALAT1 and HOTAIR in fresh-frozen LSCC samples, given their dysregulation in cancer (20). The Spearman's correlation analysis highlighted moderate dependence between HIF3α and both HIF1α and HIF2α, as well as between lncRNA MALAT1 and HOTAIR. Furthermore, a moderately strong positive tendency was observed for a linear relationship between HIF3α and lncRNA MALAT1 via Pearson's correlation analysis, indicating that a proportion of the variables could be linearly dependent. Additionally, the findings revealed a positive correlation between HIF2α and lncRNA MALAT1, which is consistent with other research (16) indicating a link between hypoxia-induced upregulation of both molecules, and their regulatory roles in metastasis and invasion. Moreover, positive associations between lncRNA MALAT1 and HIF3α transcripts have been linked to the involvement of miR-429 in cancer development (9,21–23), highlighting the complexity of the interplay between these factors in malignant transformation. Such findings underscore the potential of the HIF3α mRNA/lncRNA MALAT1/miR-429 axis as a target for therapeutic investigation in patients with LSCC and with elevated HIF3α levels. To the best of the authors' knowledge, the present study is the first to demonstrate such significant associations between lncRNAs and the major key players of angiogenesis, the HIFs.
Given that laryngeal cancer primarily develops over a protracted period and is less common in young individuals, it is notable that the present results revealed a significant association between HIF3α mRNA expression levels and age. This age-associated association may be linked to the impact of chronic hypoxia on the long-term development of laryngeal cancer. However, while the changes in HIFα expression between age groups are often contradictory across different studies, the present findings indicate the need for a more comprehensive understanding of the relationship between age and HIFα expression (24,25).
Furthermore, the present study identified a positive association between HIF3α mRNA expression levels and a positive family history of cancer. Given the major etiological factors for LSCC development in Europe (i.e., tobacco smoking and alcohol consumption), a positive family history represents a critical risk factor in malignancies. The positive family history may be related with inheritance of genetic predispositions to laryngeal cancer development or tobacco smoking addiction. The combination of both positive familial history and tobacco smoking fold increases the risk of laryngeal malignancy. The present study suggested that inherited oncological co-factors may modulate HIF3α mRNA expression, potentially contributing to an aggressive phenotype closely associated with increased HIF expression. Notably, mutations in the von Hippel-Lindau gene, a key regulator of the multiprotein complex responsible for HIF molecule degradation, have been implicated in the increased expression of HIFs in hereditary cancer (26). To the best of the authors' knowledge, the present study is the first to investigate a significant association between age, family history and HIF3α mRNA expression.
An examination of the expression of all three HIFα mRNA levels in comparison to laryngeal tumor localization yielded novel insights. A significant gradual expression of HIF1α between supraglottic, glottic and subglottic laryngeal tumors was observed, with subglottic tumors exhibiting higher HIF1α transcript levels in comparison to supraglottic and glottic tumors. While other studies have produced varying results, the present findings highlight the critical need for in-depth investigations into the role of both HIF1α mRNA and protein levels, and the mechanisms of action in LSCC (27,28). Given that tumor size is associated with increased expression levels of HIF1α, it could be hypothesized that relatively asymptomatic subglottic carcinoma may exhibit a larger extent in the same tumor stage patient subgroups, thereby demonstrating a heightened expression profile (29).
Notably, the only negative correlation was detected between HIF1α and HIF3α transcripts, a finding in line with previous studies highlighting a regulatory relationship between these two factors in different types of cancer (30,31). Moreover, the present study identified a significant positive correlation between HIF3α and HIF2α mRNAs, consistent with previous research suggesting a potential target gene relationship between HIF3α and HIF2α (32,33). The observed positive correlation may reflect a specific cellular mechanism involving alternative pathways in laryngeal cancer development, underscoring the need for further detailed investigation to clarify the monotonic correlation between HIF2α and HIF3α transcript levels.
The present study provided information on the complex interactions between HIFα subunits and hypoxia-related lncRNAs in the context of LSCC, uncovering novel associations and dependencies. The findings call for further in-depth investigations to elucidate the precise mechanisms underlying these correlations and their implications for targeted therapeutic approaches in LSCC. A major limitation of the present study includes the need for additional methodology that would confirm findings, such as immunohistochemistry and in situ hybridization.
In conclusion, the present study may improve the understanding of laryngeal cancer by demonstrating strong associations between lncRNAs and the major key players of angiogenesis, i.e. HIF isoforms. The analysis revealed a significant direct correlation between HIF3α mRNA and lncRNA MALAT1, indicating a potential axis involved in cancer cell progression under chronic hypoxia. Furthermore, the present findings highlighted novel associations between age, family history and HIF3α, and LSCC tumor location and HIF1α expression, underscoring the complex interplay between clinicopathological characteristics and HIFα forms. These results emphasize the need for further investigations to fully elucidate the intricate molecular and genetic mechanisms underlying LSCC development. The present study may stimulate new projects essential for advancing the comprehension of laryngeal cancer biology and potentially informing the development of targeted therapeutic approaches.
Acknowledgements
Not applicable.
Funding
The present study was financed by the European Union-Next Generation EU, through the National Recovery and Resilience Plan of the Republic of Bulgaria (project no. BG-RRP-2.004-0004-C01, group 3.1.5).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
SGK, GS and TMP designed the study. TMP, YH and VD performed the surgery treatments of all enrolled in the present study patients, collected the tissue samples as appropriate and information about clinicopathological features of the patients. SGK, GS and VP performed the laboratory experiments and analyzed the data. SGK and GS confirm the authenticity of all the raw data. SGK drafted the manuscript. SGK, DP, RK contributed to the data interpretation and discussion. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
Written informed consent was obtained from each patient, which is kept at the repository at the Molecular Medicine Center and the Department of ENT, Medical University, Sofia, and the current study was approved by the Ethics Committee of Medical University of Sofia (approval nos. BK-297/14.04.2015; BK-373 11.04.2016; 7836/17.11.2020 and 4584/04.07.2023).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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