CIRCULATING MICRORNA-101-3P AND MICRORNA-21-5P AS NON-INVASIVE BIOMARKERS IN THE DIAGNOSIS, RISK, PROGNOSIS AND SURVIVAL OF LARYNX CANCER

CIRCULATING MICRORNA-101-3P AND MICRORNA-21-5P AS NON-INVASIVE BIOMARKERS IN THE DIAGNOSIS, RISK, PROGNOSIS AND SURVIVAL OF LARYNX CANCER | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 26 June 2025 V1 Latest version Share on CIRCULATING MICRORNA-101-3P AND MICRORNA-21-5P AS NON-INVASIVE BIOMARKERS IN THE DIAGNOSIS, RISK, PROGNOSIS AND SURVIVAL OF LARYNX CANCER Authors : Begüm YILMAZ 0000-0001-9521-9520 [email protected] , Sinem Durmus , Haydar Murat YENER , Yetkin YILMAZ 0000-0002-5734-9751 , Irfan Papila , and Hafize Uzun Authors Info & Affiliations https://doi.org/10.22541/au.175091985.58036467/v1 185 views 114 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract ABSTRACT Introduction: To evaluate the effects of circulating microRNA-21-5q (miR-21) and microRNA-101-3p (miR-101) values on diagnosis, prognosis and survival in laryngeal squamous cell carcinoma (LSCC). Methods: Forty-six patients with LSCC and 30 healthy volunteers were included. Patients’ peripheral blood samples were analyzed for miR-21 and mir-101 and evaluated their relationship with prognostic factors for LSCC. Results: The fold change between the two groups was examined and found that miR-21 expression in the patient group was 1,708 times upregulated while miR-101 expression was found to be 0.675 times down-regulated. In the patient group, miR-21 expression with clinicalT (p=0.001), clinical stage (p=0.023), pathologicalT (p=0.002), pathological stage (p=0.006), lymphatic invasion (p=0.009) and cartilage invasion (p=0,011) was found statistically significant weak positive correlation. In the risk analysis, it was found that if miR-101 is greater than the cut-off value, it has been found to increase the disease risk by 5.20 times. Conclusions: miR-21 may be an important biomarker in the diagnosis and follow-up of LSCC. miR-101 expression can be a potential prognostic factor in LSCC. Key words: Larynx cancer, miRNA-101-3p, miRNA-21-5p, survival, prognosis Key Points Inorder to predict patient prognosis and miRs are promising novel biomarkers. There is a positive correlation of miR-21 with the prognostic features. Higher serum levels of miR-21 can predict lower five-year survival rate. miR-101 expression was found related with pN. miR-101 cut-off value raised the disease risk by 5.20 times CIRCULATING MICRORNA-101-3P AND MICRORNA-21-5P AS NON-INVASIVE BIOMARKERS IN THE DIAGNOSIS, RISK, PROGNOSIS AND SURVIVAL OF LARYNX CANCER ABSTRACT Introduction: To evaluate the effects of circulating microRNA-21-5q (miR-21) and microRNA-101-3p (miR-101) values on diagnosis, prognosis and survival in laryngeal squamous cell carcinoma (LSCC). Methods: Forty-six patients with LSCC and 30 healthy volunteers were included. Patients’ peripheral blood samples were analyzed for miR-21 and mir-101 and evaluated their relationship with prognostic factors for LSCC. Results: The fold change between the two groups was examined and found that miR-21 expression in the patient group was 1,708 times upregulated while miR-101 expression was found to be 0.675 times down-regulated. In the patient group, miR-21 expression with clinicalT (p=0.001), clinical stage (p=0.023), pathologicalT (p=0.002), pathological stage (p=0.006), lymphatic invasion (p=0.009) and cartilage invasion (p=0,011) was found statistically significant weak positive correlation. In the risk analysis, it was found that if miR-101 is greater than the cut-off value, it has been found to increase the disease risk by 5.20 times. Conclusions: miR-21 may be an important biomarker in the diagnosis and follow-up of LSCC. miR-101 expression can be a potential prognostic factor in LSCC. Key words: Larynx cancer, miRNA-101-3p, miRNA-21-5p, survival, prognosis Key Points • Inorder to predict patient prognosis and miRs are promising novel biomarkers. • There is a positive correlation of miR-21 with the prognostic features. • Higher serum levels of miR-21 can predict lower five-year survival rate. • miR-101 expression was found related with pN. • miR-101 cut-off value raised the disease risk by 5.20 times. INTRODUCTION The most common malignant tumor of the larynx is squamous cell carcinoma (SCC) and 85-95% of all laryngeal malignancies. The most important risk factors in the etiology of laryngeal cancer are tobacco and alcohol consumption. Apart from these, it is thought that genetic and familial factors related to the person and the diet of the person may also be effective in the development of cancer [1,2]. Micro RiboNucleic Acids (miRs) are non-protein-coding ribonucleic acid (RNA) molecules that form a class of endogenous small RNAs. The number of single-stranded miRs, approximately 20-23 nucleotides long, exceeds one thousand in humans. In recent years, it has been reported that the genetic causes of cancer are more complex with the demonstration that miRs are also effective in tumor formation. The genomic domains associated with cancer or the fact that more than 50% of fragile regions consist of genes encoding miR revealed that miRs are important in cancer pathogenesis [3,4]. One of the miR that is effective in carcinogenesis is miR-21, has been reported to be significantly overexpressed in LSCC tumor tissues compared to normal tissues [5]. It has been established that miR-101 functions as a tumor suppressor. Liu et al. [6] reported that miR-101 is down-regulated in LSCC tissue and is not detected in normal tissues. Whether miRs can be used in the diagnosis of laryngeal squamous cell carcinoma (LSCC) is largely unknown. The study was planned to evaluate that circulating miR-21-5q and miR-101-3p values on diagnosis, prognosis, and survival in LSCC. MATERIAL AND METHOD Study participants The study was approved by the Ethics Comittee of the XXXXX Faculty of Medicine ((Date: 3/12/2019 Issue: XXXXXXXX) and was conducted in accordance with Declaration of Helsinki. Informed written consent was obtained from all volunteers. Patients diagnosed with LSCC who sought treatment at XXX University Otorhinolaryngology & Head and Neck Surgery Department between March 2017 and September 2019. A control group was established from patients with similar demographic characteristics, without a laryngeal pathology and diagnosis of any type of malignancy. Peripheral blood samples collected from both groups were analyzed at XXX University Medical Biochemistry Department. Patient evaluation The patients were evaluated and staged based on the 2018 AJCC-HNS TNM staging groups and pathological characteristics. Statistical analysis was conducted to assess the correlation between these groups and miR levels, as well as to determine differences between the control and patient groups. Inclusion Criteria Patients; i) Individuals diagnosed with Laryngeal SCC ii) Age over 18 years old iii) Scheduled for treatment with primary surgery iv) Absence of active infection, Control group; i) Age over 18 years old. ii) No history of any malignancy. iii)No history of radiotherapy or chemotherapy. iv) Volunteering individuals without active infection. Exclusion Criteria Patients; i) Age under 18 years. ii) Pregnant or lactating iii) Additional neurological or psychiatric disease. iv) Previous history of any type of cancer. v) History of primary radiotherapy/chemotherapy and/or neoadjuvant chemotherapy. vi) Diagnosed with chronic hepatitis and/or chronic renal failure. Venous blood samples were collected into tubes after 12 hours of fasting. The samples were centrifuged for 15 minutes at 3000g. Plasma and serum were transferred to RNase/DNase‑free ependorfs and stored at −80°C until isolation. Extraction of small RNA molecules from human blood serum was performed by the EXTRACTME miRNA KIT (BLIRT, Poland) according to the manufacturer’s instructions. The RNA samples were immediately stored at -80 C, until they were reverse transcribed into cDNA. Firstly, the miR samples were transcribed into complementary DNA (cDNA) using cDNA Synthesis Kit (High Capacity) (WIZBIO SOLUTIONS, Korea). The reaction was carried out at 25 o C for 10 min, 37 o C for 120 min and 85 o C for 5 min on a StepOnePlus™ Real-Time PCR System (THERMOFISHER, USA). The cDNA samples were stored at -20 o C until used. Quantitative Real-Time PCR kits made specifically for accurate miR analysis were used to evaluate expression of the following miRNAs from serum samples: miR-21, miR-101 and RNU44 (endogenous control). For Real-Time PCR, AMPLIFYME SG Universal Mix (BLIRT, Poland) was used. The primers used in the study were obtained from SUARGE BIYOTEKNOLOJI (Istanbul, Turkey) (Table 1). Each sample was tested in triplicate on a real-time PCR system (StepOnePlus real-time PCR system, Applied Biosystems, Carlsbad, CA, USA). The RT-PCR reaction was performed at 95 oC for 10 min, followed by 40 cycles at 95 oC for 15 seconds and 60 o C for 1 min. The relative expression levels of miR-21 and miR-101 were normalized to RNU44, were calculated using the 2-ΔΔCt method [7]. Statistical analysis The suitability of values for normal distribution was assessed using the Shapiro-Wilk test. Non-parametric analysis methods were employed for values that did not meet normal distribution criteria, while parametric analysis methods were utilized for values exhibiting normal distribution. For presence-absence examinations, the chi-squared (χ2) test and Fisher’s exact test were employed. The differences in expression between the patient and control groups were compared using Student’s t-test. To compare miR expression levels between groups, the results were presented as fold changes utilizing the 2 -ΔΔCt equation [7]. The diagnostic efficacy of miR expressions was evaluated based on sensitivity, specificity, and cut-off values obtained from Receiver Operating Characteristic (ROC) curves. Risk analysis was conducted using logistic regression analysis. All analyses were carried out using the SPSS v.20 software package. A p-value <0.05 was considered statistically significant. RESULTS The mean age of the patient group was 61.2±8.8 (minimum: 44, maximum: 82), the mean age of the control group was 53.6±6.2 (minimum: 40, maximum: 64). The patient and control groups are all male. When smoking and alcohol consumption were evaluated, 100% (n = 46) were consuming cigarettes, and 19.6% (n = 9) were consuming alcohol of the patients. In the control group, 40% (n = 12) of the individuals smoking and 6.7% (n = 2) were consuming alcohol. The number of patients who smoke and drink alcohol were statistically significant compared to the control group (p <0.001 and p <0.05, respectively). The clinical characteristics and TNM stages of the patients are summarized in the Table 2. Advanced pathological features of the patient group were summarized in the Table 3. miR-21 expression levels (ΔCт) was 1,190 ± 1,439 in the patient group; while it was 1.962 ± 0.764 in the control group and a statistically significant difference was found between the two groups (p=0.010). The mean of miR-101 expression levels (ΔC т) was -1.417 ± 1.164 in the patient group; in the control group it was -1.983 ± 0.662 and a statistically significant difference was found between the two groups (p=0.009). The fold change was examined between the two groups with 2 -ΔΔ C т method, and miR-21 increased by 1,708 fold in the patient group. It was determined that miR-101 was down-regulated by 0.675 times (Fig. 1). A notable fold difference was observed in the expressions of miR-21 and miR-101. ROC curves were generated to evaluate the sensitivity and diagnostic capabilities of these miRs. In the comparison between the two groups, miR-21 exhibited a low but statistically significant AUC of 0.343 (p=0.023), while miR-101 showed an AUC of 0.694, also significantly different (p=0.005) (Fig. 2 and Table 4). When assessing LSCC risk factors, it was observed that smoking and alcohol consumption escalated the risk of the disease by 149.61 times and 8.80 times, respectively (p<0.001 and p<0.05, respectively). Cutoff values for miR-21 and miR-101 were established using the Youden index, followed by risk analysis. Subsequently, it was determined that exceeding the cut-off value for miR-21 did not significantly elevate the disease risk (p>0.05). However, surpassing the cut-off value for miR-101 amplified the disease risk by 5.20 times (95% CI: 1.76-15.39; p<0.01). Between miR-21 expression levels and Clinical T(cT) (p=0.001), Clinical Stage(cS) (p=0.023), Pathological T(pT) (p=0.002), Pathological stage(pS) (p=0.006), Lymphatic invasion (p=0.009) and cartilage invasion, a statistically significant weak positive correlation was found (p=0.011). A statistically significant weak positive correlation was found between miR-101 expression levels and miR-21 expression (p = 0.02) and pathological N(pN) (p = 0.043) in the patient group. DISCUSSION Head and neck SCC are the sixth most common cancer that includes larynx cancer [8]. Accurate TNM staging following the primary tumor diagnosis is crucial to determine the appropriate treatment approach. This may involve surgery followed by radiation therapy, chemoradiotherapy, or a combination of both, followed by salvage surgery. Despite advancements in treatment diversity, there are currently no markers available to predict patient prognosis. Following diagnosis, regular follow-ups are conducted based on the selected treatment method. Notably, individuals with similar lesions who undergo identical treatment regimens may experience differing prognoses. Presently, the underlying reasons for these differences remain unknown. Over the last decade, miRs have become an important research topic in cancer [9]. They have shown significant importance in tumor growth, invasion, angiogenesis, and immune response by controlling the expression of their target miRs [10,11]. However, the molecular mechanisms of laryngeal cancer remain unexplained. Smoking stands out as a primary risk factor for laryngeal cancer. Studies indicate that smoking influences the expression levels of numerous miRs. Dysregulation of miRs due to tobacco consumption can disrupt various mechanisms regulated by miRs, including the p53 pathway, a crucial regulator of the cell cycle, cell growth, apoptosis, and angiogenesis [12]. Notably, there was no observed correlation between smoking, alcohol use, and the expression levels of miR-21 and miR-101. The most critical prognostic factor in laryngeal cancer is the clinical stage, where advanced T and/or N stages are directly linked to decreased survival rates. Notably, the N stage holds greater predictive value for survival than the T stage. Patients with N-negative status exhibit higher survival rates than those who are N-positive. Furthermore, in cases involving cervical lymph node metastasis, the presence of histopathological extranodal invasion exacerbates the prognosis. Distant metastasis stands as the most adverse prognostic factor, with a 5-year survival rate of only 10% [13]. Ren et al. observed overexpression of miR-21 in LSCC tumor tissues, noted that downregulation of miR-21 inhibited cell proliferation and induced apoptosis in LSCC. They highlighted the correlation between increased miR-21 expression and poor differentiation, T3-4 stage, lymph node metastasis, and advanced clinical stages. These effects are likely linked to miR-21’s regulation of the Ras pathway, suggesting an oncogenic role for miR-21 in LSCC. Consequently, gene therapy targeting miR-21 is under investigation as a potential alternative strategy for treating LSCC [14]. In our study, a notable correlation was observed between miR-21 expression levels and clinical T stage, which stands as a crucial criterion in determining patient prognosis. As the T stage advances, there is a significant decrease in survival rates. Furthermore, a significant correlation was found between clinical stage groups and miR-21 expression levels. Similar relationships were noted between pT stage and pS. A weak positive correlation was observed with pN. Notably, T stage plays a pivotal role in determining the N stage; however, establishing a significant relationship might have been limited due to the small sample size in the patient group. Further analyses in larger groups are warranted. Elevated miR-21 expression levels are believed to potentially associate with LSCC risk [15]. It has been demostrasted that higher serum levels of miR-21 can predict lower five-year survival rate [16]. Considering the existing literature, the correlation between miR-21 and LSCC appears consistent. miR-101 functions as a tumor suppressor, inhibiting cell proliferation, migration, and invasion by regulating genes involved in these processes, including COX-2, EZH2, Mcl-1, Fos, Stathmin1, and c-Met [17-23]. Notably, exogenous expression of miR-101 has demonstrated reduced CDK8 expression [24]. In our study, miR-101 was significantly downregulated in the patient group. Li et al identified miR-101 was downregulated in LSCC tissue, indicated its regulation absent in normal tissues. The exogenous expression of miR-101 resulted in decreased cell proliferation, reduced invasion in the Hep-2 cell line, and decelerated xenograft tumor growth in vivo . Low levels of miR-101 expression in LSCC patients have been linked to shortened progression-free survival, consistent with existing literature [24]. Relationship between clinical data and miR-101 expression was solely observed with pN. Although pN is critical in predicting survival, it is not universally applicable to all LSCC patients, as this data is specific to patients selected for neck surgery. Despite miR-101 not showing associations with other clinical data similar to miR-21, risk analysis revealed that exceeding the miR-101 cut-off value raised the disease risk by 5.20 times. The significance of miR-101 levels in relation to general data suggests its potential as a prognostic factor. Arantes et al. [25] observed a negative association between miR-21 and treatment response to the organ preservation protocol in patients with head and neck squamous epithelial cell carcinoma (HNSCC). Multivariate analysis, adjusted for age, tumor site, p16 immunoexpression, tumor resectability, highlighted high miR-21 expression as an independent predictor of poor response to the organ preservation protocol, alongside clinical stage IV. Patients exhibiting high miR-21 expression faced poorer survival outcomes [24]. Our study’s multivariate Cox regression analysis similarly identified miR-21 and clinical stage IV as independent prognostic factors (model adjusted for age, tumor site, tumor resectability, and ’screening’ or ’validation’). These findings suggest that evaluating miR-21 expression could serve as an essential tool for treatment planning and prognostic prediction for HNSCC patients undergoing organ preservation protocols. In Li and team’s study, miR-101 exhibited downregulation in LSCC tissue, revealing its absence in normal tissues. Low miR-101 expression in LSCC patients correlated with shortened progression-free survival [24]. While numerous literature studies have explored the relationship between miR-21 and LSCC, miR-101 is a relatively novel miR in this context. Consequently, further investigations are warranted to comprehensively assess its association with LSCC. In our study, the relatively small sample size resulted in notably weak correlations between prognostic values and miR-21 and miR-101. However, the significant elevation of these two miRs in LSCC patients compared to the control group emphasizes their potential importance in LSCC, supporting their candidacy as biomarkers. Laryngeal cancer is not a homogeneous group. It is a group of cancers that has many subgroups within itself and may have different biological behavior even if they have similar characteristics. miRs, which have come to the fore in the last two decades, may answer the question of why these differences arise. There is an opinion that it can help in determining the prognosis and follow-up of patients. In this study, the positive correlation of miR-21 with the prognostic features of patients attracted much attention. It is thought that this biomarker, which has similar results in the literature, may play an important role in cancer diagnosis and follow-up in the coming years. miR-101 is a novel biomarker and its role in cancer development is not yet clear. Although it is thought that this new biomarker, which is claimed to have a more of a tumor suppressor role, may have clinical importance based on the results obtained in this study, this hypothesis needs to be retested in larger patient groups to determine this importance. Acknowledgments: Special thanks to faculty staff and members of XXXXXXX University Department of Otorhinolaryngology & Head and Neck Surgery and Medical Biochemistry. Conflicts of Interest The authors declare no conflicts of interest. REFERENCES 1. Fagan, Johannes J., et al. ”Is AJCC/UICC staging still appropriate for head and neck cancers in developing countries?.” OTO open 4.3 (2020): 2473974X20938313. 2. Barnes, L. ”Tse lly, Hunt JL, et al. Tumours of the hypopharynx, larynx and trachea.” Barnes L, Eveson JW, Reichart P, et al. World Health Organization classification of tumours: pathology and genetics of head and neck tumours. Lyon: IARC (2005): 140-145. 3. Zhang, Lin, et al. ”microRNAs exhibit high frequency genomic alterations in human cancer.” Proceedings of the National Academy of Sciences 103.24 (2006): 9136-9141. 4. Le Quesne, John, and Carlos Caldas. ”Micro-RNAs and breast cancer.” Molecular oncology 4.3 (2010): 230-241. 5. 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Arantes, Lidia Maria Rebolho Batista, et al. ”MiR-21 as prognostic biomarker in head and neck squamous cell carcinoma patients undergoing an organ preservation protocol.” Oncotarget 8.6 (2016): 9911. Supplementary Material File (figure 1.docx) Download 31.84 KB File (figure 2.docx) Download 52.09 KB File (tables.docx) Download 20.84 KB Information & Authors Information Version history V1 Version 1 26 June 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Begüm YILMAZ 0000-0001-9521-9520 [email protected] TC Saglik Bakanligi Basaksehir Cam ve Sakura Sehir Hastanesi View all articles by this author Sinem Durmus Izmir Katip Celebi Universitesi Tip Fakultesi View all articles by this author Haydar Murat YENER Istanbul Universitesi-Cerrahpasa Cerrahpasa Tip Fakultesi View all articles by this author Yetkin YILMAZ 0000-0002-5734-9751 Istanbul Universitesi-Cerrahpasa Cerrahpasa Tip Fakultesi View all articles by this author Irfan Papila Istanbul Universitesi-Cerrahpasa Cerrahpasa Tip Fakultesi View all articles by this author Hafize Uzun Istanbul Atlas Universitesi Tip Fakultesi View all articles by this author Metrics & Citations Metrics Article Usage 185 views 114 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Begüm YILMAZ, Sinem Durmus, Haydar Murat YENER, et al. 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