Diagnostic Potential of miR-221, miR-214, and miR-375 Expression in Odontogenic Keratocysts Versus Dentigerous Cysts: An In Vitro Comparison

Diagnostic Potential of miR-221, miR-214, and miR-375 Expression in Odontogenic Keratocysts Versus Dentigerous Cysts: An In Vitro Comparison | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diagnostic Potential of miR-221, miR-214, and miR-375 Expression in Odontogenic Keratocysts Versus Dentigerous Cysts: An In Vitro Comparison Sareh Farhadi, Maliheh Entezari, Dorsa Abdi, Salar Mirzaei, Sina Noruozian Fard, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7298969/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in BMC Oral Health → Version 1 posted 15 You are reading this latest preprint version Abstract Background : The early diagnosis of odontogenic cysts is vital to prevent future complications and choosing an effective treatment plan. Dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) are two common developmental cysts that affect the jaw. OKCs show locally aggressive behavior and a higher recurrence rate compared with DCs, emphasizing the need for reliable molecular diagnostic markers. MicroRNAs (miRNAs) are abnormally expressed in tumors and lesions with aggressive behavior and can be used as diagnostic biomarkers. This study investigates the diagnostic potential of miR-221, miR-214, and miR-375 expression in OKCs and DCs, due to the invasive nature of OKCs compared with DCs. Materials and Methods : This study analyzed 36 paraffin-embedded odontogenic cyst tissue samples (23 OKC and 13 DC) collected from the oral pathology archive of Islamic Azad University. Total RNA, including miRNAs, was extracted using TRIzol-based protocols with RNase-free conditions. RNA purity and integrity were assessed via spectrophotometry and agarose gel electrophoresis. Reverse transcription was performed using the M-MLV RNase H kit. Specific primers for miR-221, miR-214, and miR-375 were designed and verified. Quantitative real-time PCR was conducted using SYBR Green, and delta cycle threshold (ΔCt) values were calculated. Results : miR-221, miR-214, and miR-375 showed significantly lower ΔCt values in OKCs compared with DCs (p < 0.001). The mean ΔCt values for miR-221, miR-214, and miR-375 in OKCs were 11.27, 10.10, and 8.18, respectively, compared with 12.95, 12.51, and 11.05 in DCs. Conclusion : The ΔCt values for miR-221, miR-214, and miR-375s were significantly lower in OKC samples, indicating higher expression levels compared with DCs.The significant upregulation of these miRNAs in OKCs compared with DCs suggests that these miRNAs have potential as diagnostic biomarkers. Odontogenic keratocyst Dentigerous cyst MicroRNA miR-221 miR-214 miR-375 Figures Figure 1 Introduction Odontogenic cysts and jaw osseous lesions are not generally encountered in the routine practice of many dentists. However, they can lead to bony destruction, resorption, or displacement of adjacent teeth, and even dysplasia or malignant transformation, posing challenges in diagnosis and treatment ( 1 ). Dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) are two common developmental cysts affecting the jaw. DCs are related to impacted teeth and represent the second most frequent jaw cysts; they represent 14%-20% of all jaw cysts ( 2 , 3 ). DCs are mostly asymptomatic and are typically discovered incidentally during the evaluation of unerupted teeth ( 4 ). When symptoms do occur, they usually include pain and bone expansion. They are frequently located near the lower third molars, upper canines, lower premolars, and upper third molars. These cysts may cause delayed tooth eruption, missing teeth, or abnormal tooth positioning within the dental arch ( 5 , 6 ). In the absence of treatment, the progression of the cyst remains localized, with destruction and expansion limited to peripheral structures. Malignant transformation is extremely rare( 7 ). OKCs are benign lesions in the jaw of odontogenic origin and make up 10% of all jaw cysts. About 75% of these cysts occur in the mandible, mainly in the molar and ramus regions ( 7 ). In the 2005 WHO classification, OKCs were labeled as keratocystic odontogenic tumors (KOTs) because of their aggressive behavior and relatively high recurrence rate ( 8 ). However, the evidence for neoplastic activity was insufficient to keep their tumor classification, and the term “KOT” was changed back to its original name, “OKC” ( 9 ). Long-standing OKCs can potentially undergo malignant transformation, especially due to persistent chronic inflammation. Epithelial dysplasia is a diagnostic challenge because of variability in its detection and grading by different examiners and even by the same examiner at different times. The development of squamous cell carcinoma (SCC) from an OKC is not well understood. Early detection of dysplasia is crucial for effective treatment and a better prognosis ( 10 ). MicroRNAs (miRNAs) are a family of small, conserved, non-coding RNA molecules, approximately 18 to 24 nucleotides long. They are involved in the post-transcriptional regulation of genes ( 7 , 8 ). More than 60% of all mammalian genes are predicted to be regulated by these molecules ( 9 ). Previous research has shown that miRNAs play a fundamental role in cancer development and progression, including differentiation, proliferation, invasion, migration, and angiogenesis. Their expression often becomes dysregulated across various cancer types, with functional differences depending on the tissue type ( 10 , 11 ). Among the deregulated microRNAs, miR-221 is known for its oncogenic role and potential as a biomarker, as it suppresses tumor suppressor genes ( 12 ). MiR-375 is also frequently downregulated in several cancer types; it acts as a tumor suppressor and inhibits malignant cell properties ( 13 ). MiR-214 is another tumor suppressor microRNA that is essential for regulating tumor invasiveness, proliferation, metastasis, angiogenesis, and resistance to chemotherapy ( 14 ). Studies have shown that OKCs and DCs can be potentially premalignant lesions, and some miRNAs may play a role in this transformation. Despite the importance of miRNA, its role in epithelial odontogenic cysts and tumors is not well documented. Considering the characteristic behavior of OKC in comparison to other odontogenic cysts and the high prevalence of DC, the present study aims to compare the expression of mir-221, mir-214, and mir-375 in DCs and OKCs ( 7 ). To the best of our knowledge, this topic has not been previously investigated in odontogenic cysts, whereas previous research has been limited to head and neck cancers, predominantly SCC ( 8 – 11 ). Materials and methods The study was approved by the institutional research ethics committee (IR.IAU.DENTAL.REC.1401.033) Human tissue specimens This in vitro study was conducted using formalin-fixed, paraffin-embedded (FFPE) tissue specimens to evaluate the expression of selected miRNAs. A total of 36 tissue samples (23 OKC and 13 DC specimens) were collected from the oral pathology archive at the Islamic Azad University, Tehran Medical Branch. Histopathological analysis confirmed all samples as either OKCs or DCs. The preservation conditions and tissue fixation processes were acceptable. RNA extraction The miRNA molecule is chemically more reactive than DNA and is rapidly degraded by RNase enzymes. Handling miRNA requires greater precision compared with DNA, hence, the use of RNase-free materials and tools is necessary. To ensure this, all containers and tubes used in miRNA extraction were autoclaved at 15 psi for 15 minutes. Oral biopsy samples, preserved in formalin and embedded in paraffin, were analyzed. Total RNA extraction kit (Parstous, Iran) was used to extract Total RNA from the isolated cells. Tissue samples were crushed and immersed in 1.5 ml of TRIzol solution. The crushed tissue was placed in a microtube, lysed with 1 mL of Riboex solution, and then mixed with 0.2 mL of chloroform. The mixture was vortexed at high speed for 15 seconds before centrifugation at 12,000 × g for 15 minutes at 4°C. The upper aqueous phase was carefully collected, mixed with 0.4 ml of isopropanol, and vortexed again for 15 seconds. The mixture was inverted 2–3 times to enhance sediment formation and held at -20°C for over 12 hours. The samples were incubated at 25°C for 10 minutes before centrifugation at 12,000 × g at 4°C. A white RNA pellet formed, followed by a clear supernatant removed to isolate the sediment. The RNA pellets were washed with 75% ethanol twice before air-drying. The RNA pellets were then dissolved in 0.05 ml of diethylpyrocarbonate (DEPC)-treated water for further analysis ( 12 , 13 ). RNA quantification and integrity control The RNA concentration was determined using UV spectrophotometry (Amersham NanoVue Plus, USA), and its integrity was confirmed using 1% agarose gel electrophoresis. For more precise assessment of RNA integrity, an electropherogram was utilized to calculate the 28S/18S ribosomal RNA ratio. Samples with 28S/18S ratios greater than 1.4, indicating high RNA quality, were deemed suitable for downstream analysis. A Nanodrop spectrophotometer (Thermo Scientific, USA) was used to quantify the RNA at 260 nm. Optical density (OD) was assessed at four specific wavelengths: 230 nm (contaminant and background absorption), 260 nm (maximum absorption for nucleic acids and proteins), 280 nm (contaminant and background absorption), and 320 nm. The ratios OD260/230 and OD260/280 were analyzed to evaluate RNA purity ( 14 ). Reverse transcription The M-MLV RNase H- kit (BioFact, Daejeon, Republic of Korea) was used to reverse-transcribe total RNA into complementary DNA. To denature the RNA and facilitate primer annealing, dilute whole RNA to 1000 ng/uL, add 1 µL of random hexamer, and incubate at 65°C for 5 minutes. Then immediately placed on ice to cool down (to avoid premature enzyme activation). After adding 10 µL of RT Master Mix enzyme, the plate was inserted in the Gradient Cycler (Razi Tajhiz, Tehran, Iran). The temperature protocol was started: 25°C / 10 min (primer connection), 42°C /60 min (cDNA synthesis) 70°C / 10 min (enzyme inactivation) and 4°C/ 10 min (maintenance)( 15 ). Primer design Specific primers for miRNA analysis were designed using Oligo Analyzer and Primer3 Plus software. Primer sequences were verified using BLAST, and primers were synthesized by ParsGenome (Tehran, Iran) under code PG4487-03. Real‑time polymerase chain reaction SYBR Green dye (TaKaRa, Otsu, Shiga, Japan) was used to perform Quantitative PCR (qPCR). First, a master mix containing water, primers, and Sybr Green was created for the number of reactions, followed by an additional sample (as a negative control) to execute many reactions in parallel and limit the possibility of pipetting errors. After a gentle spin, the master mix was divided into separate tubes containing 18 µl. Then, 2 µl of cDNA was added to each tube and placed in the apparatus. Each sample was taken twice. The qPCR process was initiated: pre-denaturation (95°C/15 min), denaturation (95°C/15 S), annealing (60°C/30 S), and extension (72°C/20 S), with the primer sequences listed in Table 1 . Table 1 Sequences of the primers. microRNA symbol Primer Sequence miR-221 Forward5′-ACUGGCAUACAAUGUAGAUUU-3′ Reverse5′-AGCUACAUUGUCUGCUGGGUUUC-3′ miR-214 forward 5'-TGCGGACAGCAGGCACAGAC-3' Reverse 5'-CCAGTGCAGGGTCCGAGGT-3' miR-375 forward 5'-GCCCGCTTTGTTCGTTCGGCT-3' Reverse 5'-GTGCAGGGTCCGAGGT-3' After completing the preceding processes, a melting curve analysis was conducted after the qPCR process to evaluate the specificity of the amplified products. The curves were carefully examined to detect any signs of non-specific amplification or primer-dimer formation. Samples that showed abnormal melting behavior were marked for further review, and only those exhibiting a single, sharp peak were considered suitable and included in the final analysis. Finally, the relative expression level of miRNAs was determined using the amplification cycle threshold (Ct) ( 15 – 17 ). The relative expression levels of miRNAs were determined using the Ct values. ( 18 ). ΔCt values were calculated using the formula: ΔCt = Ct(gene) − Ct(housekeeping gene) Relative Expression = 2^(−ΔCt) ΔΔCt = ΔCt(treated) − average ΔCt(control) Fold Change = 2^(−ΔΔCt) Statistical analysis SPSS software, version 29.0 (IBM Corp., Armonk, NY, USA) was used to conduct statistical analyses. A p-value of < 0.05 was considered statistically significant. Statistical analyses were performed separately for each of the three microRNAs. The Kolmogorov-Smirnov test evaluated data normality. Normally distributed data were analyzed using an Independent Sample t-test, whereas non-normal data were analyzed using the Mann-Whitney U. Results In total, 36 FFPE tissue samples were analyzed, including 23 OKCs and 13 DCs. QPCR was performed to evaluate the expression of miR-221, miR-214, and miR-375. Melt curve analysis confirmed the specificity of the amplifications, and all reactions were carried out in triplicate. The data are presented as mean ± standard deviation (SD). The independent t-test revealed statistically significant differences in the ΔCt values of all three miRNAs between OKC and DC groups (p < 0.001 for each comparison), indicating higher expression in OKCs. As shown in Table 2 , the mean ΔCt value for miR-221 in OKCs was 11.27 ± 0.91, which was significantly lower than 12.95 ± 0.66 in DCs (p < 0.001). According to Table 3 , the mean ΔCt value of miR-214 was 10.10 ± 0.61 in OKCs and 12.51 ± 0.46 in DCs (p < 0.001). Similarly, Table 4 shows that miR-375 had a mean ΔCt of 8.18 ± 0.63 in OKCs versus 11.05 ± 0.86 in DCs (p < 0.001). Table 2 Expression Differences of miR-221 between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values. Mean ΔCT Standard deviation lowest highest P value OKC 11.2691 0.90787 9.87 12.93 < 0.001 DC 12.9523 0.65736 11.69 14.09 < 0.001 Table 3 Expression Differences of miR-214 between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values. Mean ΔCT Standard deviation lowest highest P value OKC 10.0978 0.60916 9.39 11.42 < 0.001 DC 12.5091 0.45686 11.89 13.34 < 0.001 Table 4 Expression Differences of miR-375 between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values. Mean ΔCT Standard deviation lowest highest P value OKC 8.1803 0.63364 6.83 9.69 < 0.001 DC 11.0542 0.86091 9.80 12.54 < 0.001 These ΔCt differences were further translated into relative expression values and fold changes using the 2^(-ΔΔCt) method. As summarized in Table 5 , the fold change of miR-221, miR-214, and miR-375 in OKC tissues was markedly higher than in DC tissues. Figure 1 graphically illustrates the differences in ΔCt values between the two groups. Table 5 The calculated relative expression levels and fold changes (Odontogenic keratocyst vs dentigerous cyst). Relative Expression in OKC Relative Expression in DC Fold Change (OKC /DC) miR-221 0.000405 0.000126 3.20 miR-214 0.000911 0.000171 5.31 miR-375 0.003448 0.000472 7.31 Discussion We investigated the diagnostic potential of miR-221, miR-214, and miR-375 expression in OKCs and DCs, due to aggressive growth and higher recurrence rates of OKCs compared with DCs. Our results demonstrate that the expression levels of all three miRNAs in OKCs are significantly elevated in OKC samples relative to DCs. This differential expression suggests that these miRNAs may serve as valuable molecular markers for distinguishing between these two common types of odontogenic cysts. Furthermore, upregulation of certain miRNAs in OKCs may reflect underlying molecular pathways associated with the more proliferative and invasive nature of this lesion. To the best of our knowledge, this is the first study to investigate the expression of these specific miRNAs in odontogenic cysts, highlighting novelty and potential contribution to the field. Epithelial odontogenic cysts and tumors originate from the odontogenic epithelium and the surface epithelial lining of the oral mucosa. The difference in the proliferation of epithelial cells has an important role in the pathogenesis of these lesions ( 19 ). The behavior of a lesion is generally associated with its growth potential, which can be assessed by measuring cellular proliferative activity. This growth potential and its correlation with clinical aggressiveness and recurrence can determine the lesion's nature. Experimental studies indicate that the extent of cellular proliferation within a tumor can serve as an indicator of its biological aggression ( 20 ). Studies have shown that OKCs and DCs can be potential premalignant lesions, and some miRNAs may play a role in this transformation. The change in the expression of these miRNAs is necessary for the initial phases of cancer development. It should be considered evidence of the existence of the potential for malignant transformation of a lesion. Several studies have reported that most of the identified miRNA genes are located within cancer-associated genomic regions, often referred to as fragile sites. As such, identifying these miRNAs may aid in differential diagnosis and assessing malignant potential ( 11 , 21 – 23 ). Understanding the role of miRNA in cancer biology is challenging because of its biphasic nature, where the same miRNA can function as an oncomir in one cancer type and as a tumor suppressor in another. For example, miRNA-29 acts as an oncogene in breast cancer but serves as a tumor suppressor in lung cancer. This dual role can be attributed to miRNAs targeting different messenger RNAs (mRNAs) in various tissues, often through diverse pathways ( 24 ). According to past studies, miR-221, miR-214, and miR-375 are not only heavily dysregulated, but also their expression levels in cancer tissues were completely variable ( 2 , 25 , 26 ). Recent research has used miRNAs or specific miRNA ratios as prognostic markers in head and neck squamous cell carcinoma. For example, the ratio of miR-221 to miR-375 was highly specific (0.93) and sensitive (0.92) for distinguishing normal from malignant tissues ( 27 ). MiRNA-221 is part of the miRNA-221/222 cluster encoded in chromosome Xp11.3 ( 27 ), and it has dual roles in cancer as an oncomiR or tumor suppressor ( 28 ). Overexpression of miR-221 has been linked to increased proliferation in glioblastoma ( 12 ), gastric cancer ( 29 ), renal cell carcinoma ( 30 ), prostate cancer ( 31 ), and oral squamous cell carcinoma ( 32 ). However, Xie et al. reported that miR-221 inhibits pancreatic cancer cell proliferation through the JAK-STAT3 pathway ( 33 ). It has also been shown that miRNA-222 regulates invasion in tongue squamous cell carcinoma by targeting specific enzymes ( 34 ). Changes in miRNA expression impact signaling pathways and can lead to carcinogenesis and aggressive behavior ( 35 ). MiR-214 is located on chromosome 1q24.3 ( 28 ), and it was initially identified for its ability to induce apoptosis in HeLa cells by inhibiting the anti-apoptotic protein Bcl2l2 and enhancing pro-apoptotic factors like Bax and caspases ( 29 ). Additionally, it targets oncogenic mRNAs, including CD44 and CDK1, and leads to reduced proliferation and migration in various cancers ( 30 , 31 ). MiR-214 exhibits variable expression across cancer types and functions as both a tumor suppressor and an oncogenic miRNA. In Oral Squamous Cell Carcinoma (OSCC), it induces malignancy through the MAPK/ERK pathway and correlates with poor prognosis ( 16 ), while its inhibition enhances apoptosis ( 32 ). A miR-based prognostic model including miR-214-3p appeared to be effective for the detection of early-stage OSCC ( 11 ). MiR-375 is located on chromosome 2 and plays tissue-specific roles as both a tumor suppressor and an onco-miRNA ( 33 , 34 ). It suppresses malignancy in nasopharyngeal carcinoma ( 35 ) but promotes breast cancer progression by targeting HOXA5 ( 36 ). In oral potentially malignant disorders (OPMD), salivary miR-375 levels are lower in patients with dysplasia, which indicates its potential as a sensitive marker ( 37 ). In OSCC, downregulation of miR-375 correlates with poor prognosis, reduced survival, and lymph node metastasis ( 38 ). Elevated miR-375 levels inhibit proliferation and invasion by targeting p53 and SLC7A11, enhancing apoptosis and radiosensitivity ( 39 ). Based on the findings of this study and considering that specific miRNAs exhibit high tissue and organ specificity, it seems that assessing the expression of particular miRNAs could be valuable for predicting the clinical behavior of odontogenic cysts. Currently, techniques such as qRT-PCR and microarray are used for miRNA profiling, which are non-invasive, cost-effective, and reliable ( 40 ). However, several challenges remain when using miRNAs, such as the complex nature of miRNA-mRNA interactions. A single miRNA can target multiple mRNAs and vice versa. Therefore, standardizing or normalizing miRNA levels in experimental studies of these interactions requires extensive research, which can be challenging. Fortunately, bioinformatics tools and algorithms are available to predict and assess miRNA-mRNA interactions based on sequence data ( 23 ). Studies from various countries have reported variations in microRNA expression in oral cancer patients, raising concerns about the reliability of miRNAs as biomarkers for cancer. For example, miR-21 levels were higher in plasma from the Japanese and Danish cohorts, but lower in the Turkish cohort and unchanged in Chinese patients ( 41 ). Consequently, further studies are necessary to provide a clearer explanation. Another limitation of this study was the use of FFPE tissue to extract miRNAs. Since tissue lysis can alter miRNA concentrations, the processing procedure may affect the outcome ( 42 ). This emphasizes the need for additional investigations utilizing fresh tissue samples. The differential expression of miR-221, miR-214, and miR-375 suggests their potential utility as molecular diagnostic tools. Their application in clinical diagnostics could facilitate early detection and management of odontogenic cysts with aggressive behavior, reducing recurrence and unnecessary extensive surgical interventions. Conclusion In summary, a significant difference in the expression levels of miR-221, miR-214, and miR-375 was observed between OKCs and DCs. The significant downregulation of these miRNAs in DCs compared with OKCs indicates their potential as diagnostic biomarkers. Therefore, monitoring the expression levels of miRNAs could be used as a valuable tool for the diagnosis and prognosis of odontogenic cysts. These findings provide insights into the molecular differences between OKCs and DCs and facilitate a deeper understanding and evaluation of their clinical behavior. Declarations Conflict of Interest The Authors confirm that they have no conflict of interest. Funding None. 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Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in BMC Oral Health → Version 1 posted Editorial decision: Revision requested 15 Sep, 2025 Reviews received at journal 14 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviews received at journal 12 Sep, 2025 Reviews received at journal 07 Sep, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 24 Aug, 2025 Reviewers agreed at journal 24 Aug, 2025 Reviewers agreed at journal 24 Aug, 2025 Reviewers agreed at journal 23 Aug, 2025 Reviewers invited by journal 22 Aug, 2025 Editor invited by journal 13 Aug, 2025 Editor assigned by journal 13 Aug, 2025 Submission checks completed at journal 13 Aug, 2025 First submitted to journal 05 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7298969","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506219804,"identity":"6b6d64be-3812-4ac4-a72b-5cacf2e20337","order_by":0,"name":"Sareh Farhadi","email":"","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Sareh","middleName":"","lastName":"Farhadi","suffix":""},{"id":506219805,"identity":"8a27658a-4a84-41d2-8589-3c1f7fbfb5ef","order_by":1,"name":"Maliheh Entezari","email":"","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Maliheh","middleName":"","lastName":"Entezari","suffix":""},{"id":506219806,"identity":"d5a87823-0feb-4f20-bff2-17d10c20d58b","order_by":2,"name":"Dorsa Abdi","email":"","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Dorsa","middleName":"","lastName":"Abdi","suffix":""},{"id":506219807,"identity":"9c003525-0879-4565-8f4c-b3ea015f176d","order_by":3,"name":"Salar Mirzaei","email":"","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Salar","middleName":"","lastName":"Mirzaei","suffix":""},{"id":506219808,"identity":"c370b7a6-ee58-4408-9dc1-1efd556c6002","order_by":4,"name":"Sina Noruozian Fard","email":"","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Sina","middleName":"Noruozian","lastName":"Fard","suffix":""},{"id":506219809,"identity":"05e8dd46-2107-484e-a94f-635fc744cc86","order_by":5,"name":"Sana Baghizadeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYJACZiBOYGNvAFIGFqRo4TkA0iJBghYGiQQQmwgt8u1nD34uqKnN45N8fnXDjwIJBv727gS8WgzO5CVLzzh2vJhNOqfsZg/QYRJnzm7Ar4Uhx0Cah+1YYpt0TtoNHqAWA4lc/Frk+98Y/+b5B9QieSbt5h9itDDcyDGT5m2rSWyTYD92myhbDG68MbOe2XcgsY0nh+22jIEED0G/yPfnGN8u+FaXOL/9+LObb/7YyPG39xJwGAQcBmIeAxCLhxjlIFAHxOwPiFU9CkbBKBgFIwwAAPDHR1vFcLRgAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic Azad University Medical Branch of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Sana","middleName":"","lastName":"Baghizadeh","suffix":""}],"badges":[],"createdAt":"2025-08-05 09:38:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7298969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7298969/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-025-07289-0","type":"published","date":"2025-12-12T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90328103,"identity":"315e4ab8-6970-4411-a0c8-97e4b0f53fb4","added_by":"auto","created_at":"2025-09-01 12:33:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28765,"visible":true,"origin":"","legend":"\u003cp\u003eExpression differences of miRNAs between odontogenic keratocyst (OKC) and dentigerous cyst (DC) based on ΔCt values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7298969/v1/30549b87d770565fb1960ce2.png"},{"id":98244139,"identity":"3e964c83-7bb2-4a65-b71d-cdf551ce37d2","added_by":"auto","created_at":"2025-12-15 16:13:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":699107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7298969/v1/e279fcd4-495d-4554-a2eb-0041a90ba422.pdf"},{"id":90328108,"identity":"268afa94-3a30-4315-ad48-d2b0b80560ea","added_by":"auto","created_at":"2025-09-01 12:33:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3349356,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7298969/v1/7f74035d67ddf327bea25246.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diagnostic Potential of miR-221, miR-214, and miR-375 Expression in Odontogenic Keratocysts Versus Dentigerous Cysts: An In Vitro Comparison","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOdontogenic cysts and jaw osseous lesions are not generally encountered in the routine practice of many dentists. However, they can lead to bony destruction, resorption, or displacement of adjacent teeth, and even dysplasia or malignant transformation, posing challenges in diagnosis and treatment (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) are two common developmental cysts affecting the jaw. DCs are related to impacted teeth and represent the second most frequent jaw cysts; they represent 14%-20% of all jaw cysts (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). DCs are mostly asymptomatic and are typically discovered incidentally during the evaluation of unerupted teeth (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). When symptoms do occur, they usually include pain and bone expansion. They are frequently located near the lower third molars, upper canines, lower premolars, and upper third molars. These cysts may cause delayed tooth eruption, missing teeth, or abnormal tooth positioning within the dental arch (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In the absence of treatment, the progression of the cyst remains localized, with destruction and expansion limited to peripheral structures. Malignant transformation is extremely rare(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). OKCs are benign lesions in the jaw of odontogenic origin and make up 10% of all jaw cysts. About 75% of these cysts occur in the mandible, mainly in the molar and ramus regions (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In the 2005 WHO classification, OKCs were labeled as keratocystic odontogenic tumors (KOTs) because of their aggressive behavior and relatively high recurrence rate (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, the evidence for neoplastic activity was insufficient to keep their tumor classification, and the term \u0026ldquo;KOT\u0026rdquo; was changed back to its original name, \u0026ldquo;OKC\u0026rdquo; (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Long-standing OKCs can potentially undergo malignant transformation, especially due to persistent chronic inflammation. Epithelial dysplasia is a diagnostic challenge because of variability in its detection and grading by different examiners and even by the same examiner at different times. The development of squamous cell carcinoma (SCC) from an OKC is not well understood. Early detection of dysplasia is crucial for effective treatment and a better prognosis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs) are a family of small, conserved, non-coding RNA molecules, approximately 18 to 24 nucleotides long. They are involved in the post-transcriptional regulation of genes (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). More than 60% of all mammalian genes are predicted to be regulated by these molecules (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Previous research has shown that miRNAs play a fundamental role in cancer development and progression, including differentiation, proliferation, invasion, migration, and angiogenesis. Their expression often becomes dysregulated across various cancer types, with functional differences depending on the tissue type (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Among the deregulated microRNAs, miR-221 is known for its oncogenic role and potential as a biomarker, as it suppresses tumor suppressor genes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). MiR-375 is also frequently downregulated in several cancer types; it acts as a tumor suppressor and inhibits malignant cell properties (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). MiR-214 is another tumor suppressor microRNA that is essential for regulating tumor invasiveness, proliferation, metastasis, angiogenesis, and resistance to chemotherapy (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStudies have shown that OKCs and DCs can be potentially premalignant lesions, and some miRNAs may play a role in this transformation. Despite the importance of miRNA, its role in epithelial odontogenic cysts and tumors is not well documented. Considering the characteristic behavior of OKC in comparison to other odontogenic cysts and the high prevalence of DC, the present study aims to compare the expression of mir-221, mir-214, and mir-375 in DCs and OKCs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). To the best of our knowledge, this topic has not been previously investigated in odontogenic cysts, whereas previous research has been limited to head and neck cancers, predominantly SCC (\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe study was approved by the institutional research ethics committee (IR.IAU.DENTAL.REC.1401.033)\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuman tissue specimens\u003c/h2\u003e\u003cp\u003eThis in vitro study was conducted using formalin-fixed, paraffin-embedded (FFPE) tissue specimens to evaluate the expression of selected miRNAs. A total of 36 tissue samples (23 OKC and 13 DC specimens) were collected from the oral pathology archive at the Islamic Azad University, Tehran Medical Branch. Histopathological analysis confirmed all samples as either OKCs or DCs. The preservation conditions and tissue fixation processes were acceptable.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA extraction\u003c/h3\u003e\n\u003cp\u003eThe miRNA molecule is chemically more reactive than DNA and is rapidly degraded by RNase enzymes. Handling miRNA requires greater precision compared with DNA, hence, the use of RNase-free materials and tools is necessary. To ensure this, all containers and tubes used in miRNA extraction were autoclaved at 15 psi for 15 minutes. Oral biopsy samples, preserved in formalin and embedded in paraffin, were analyzed. Total RNA extraction kit (Parstous, Iran) was used to extract Total RNA from the isolated cells. Tissue samples were crushed and immersed in 1.5 ml of TRIzol solution. The crushed tissue was placed in a microtube, lysed with 1 mL of Riboex solution, and then mixed with 0.2 mL of chloroform. The mixture was vortexed at high speed for 15 seconds before centrifugation at 12,000 \u0026times; g for 15 minutes at 4\u0026deg;C. The upper aqueous phase was carefully collected, mixed with 0.4 ml of isopropanol, and vortexed again for 15 seconds. The mixture was inverted 2\u0026ndash;3 times to enhance sediment formation and held at -20\u0026deg;C for over 12 hours. The samples were incubated at 25\u0026deg;C for 10 minutes before centrifugation at 12,000 \u0026times; g at 4\u0026deg;C. A white RNA pellet formed, followed by a clear supernatant removed to isolate the sediment. The RNA pellets were washed with 75% ethanol twice before air-drying. The RNA pellets were then dissolved in 0.05 ml of diethylpyrocarbonate (DEPC)-treated water for further analysis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA quantification and integrity control\u003c/h3\u003e\n\u003cp\u003eThe RNA concentration was determined using UV spectrophotometry (Amersham NanoVue Plus, USA), and its integrity was confirmed using 1% agarose gel electrophoresis. For more precise assessment of RNA integrity, an electropherogram was utilized to calculate the 28S/18S ribosomal RNA ratio. Samples with 28S/18S ratios greater than 1.4, indicating high RNA quality, were deemed suitable for downstream analysis. A Nanodrop spectrophotometer (Thermo Scientific, USA) was used to quantify the RNA at 260 nm. Optical density (OD) was assessed at four specific wavelengths: 230 nm (contaminant and background absorption), 260 nm (maximum absorption for nucleic acids and proteins), 280 nm (contaminant and background absorption), and 320 nm. The ratios OD260/230 and OD260/280 were analyzed to evaluate RNA purity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eReverse transcription\u003c/h3\u003e\n\u003cp\u003eThe M-MLV RNase H- kit (BioFact, Daejeon, Republic of Korea) was used to reverse-transcribe total RNA into complementary DNA. To denature the RNA and facilitate primer annealing, dilute whole RNA to 1000 ng/uL, add 1 \u0026micro;L of random hexamer, and incubate at 65\u0026deg;C for 5 minutes. Then immediately placed on ice to cool down (to avoid premature enzyme activation). After adding 10 \u0026micro;L of RT Master Mix enzyme, the plate was inserted in the Gradient Cycler (Razi Tajhiz, Tehran, Iran). The temperature protocol was started: 25\u0026deg;C / 10 min (primer connection), 42\u0026deg;C /60 min (cDNA synthesis) 70\u0026deg;C / 10 min (enzyme inactivation) and 4\u0026deg;C/ 10 min (maintenance)(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePrimer design\u003c/h3\u003e\n\u003cp\u003eSpecific primers for miRNA analysis were designed using Oligo Analyzer and Primer3 Plus software. Primer sequences were verified using BLAST, and primers were synthesized by ParsGenome (Tehran, Iran) under code PG4487-03.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eReal‑time polymerase chain reaction\u003c/h2\u003e\u003cp\u003eSYBR Green dye (TaKaRa, Otsu, Shiga, Japan) was used to perform Quantitative PCR (qPCR). First, a master mix containing water, primers, and Sybr Green was created for the number of reactions, followed by an additional sample (as a negative control) to execute many reactions in parallel and limit the possibility of pipetting errors. After a gentle spin, the master mix was divided into separate tubes containing 18 \u0026micro;l. Then, 2 \u0026micro;l of cDNA was added to each tube and placed in the apparatus. Each sample was taken twice. The qPCR process was initiated: pre-denaturation (95\u0026deg;C/15 min), denaturation (95\u0026deg;C/15 S), annealing (60\u0026deg;C/30 S), and extension (72\u0026deg;C/20 S), with the primer sequences listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequences of the primers.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003emicroRNA symbol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer Sequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward5\u0026prime;-ACUGGCAUACAAUGUAGAUUU-3\u0026prime;\u003c/p\u003e\u003cp\u003eReverse5\u0026prime;-AGCUACAUUGUCUGCUGGGUUUC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-214\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward 5'-TGCGGACAGCAGGCACAGAC-3'\u003c/p\u003e\u003cp\u003eReverse 5'-CCAGTGCAGGGTCCGAGGT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eforward 5'-GCCCGCTTTGTTCGTTCGGCT-3'\u003c/p\u003e\u003cp\u003eReverse 5'-GTGCAGGGTCCGAGGT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAfter completing the preceding processes, a melting curve analysis was conducted after the qPCR process to evaluate the specificity of the amplified products. The curves were carefully examined to detect any signs of non-specific amplification or primer-dimer formation. Samples that showed abnormal melting behavior were marked for further review, and only those exhibiting a single, sharp peak were considered suitable and included in the final analysis. Finally, the relative expression level of miRNAs was determined using the amplification cycle threshold (Ct) (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The relative expression levels of miRNAs were determined using the Ct values. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). ΔCt values were calculated using the formula:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eΔCt\u0026thinsp;=\u0026thinsp;Ct(gene)\u0026thinsp;\u0026minus;\u0026thinsp;Ct(housekeeping gene)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eRelative Expression\u0026thinsp;=\u0026thinsp;2^(\u0026minus;ΔCt)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eΔΔCt\u0026thinsp;=\u0026thinsp;ΔCt(treated)\u0026thinsp;\u0026minus;\u0026thinsp;average ΔCt(control)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eFold Change\u0026thinsp;=\u0026thinsp;2^(\u0026minus;ΔΔCt)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eSPSS software, version 29.0 (IBM Corp., Armonk, NY, USA) was used to conduct statistical analyses. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were performed separately for each of the three microRNAs. The Kolmogorov-Smirnov test evaluated data normality. Normally distributed data were analyzed using an Independent Sample t-test, whereas non-normal data were analyzed using the Mann-Whitney U.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn total, 36 FFPE tissue samples were analyzed, including 23 OKCs and 13 DCs. QPCR was performed to evaluate the expression of miR-221, miR-214, and miR-375. Melt curve analysis confirmed the specificity of the amplifications, and all reactions were carried out in triplicate. The data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e\u003cp\u003eThe independent t-test revealed statistically significant differences in the ΔCt values of all three miRNAs between OKC and DC groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for each comparison), indicating higher expression in OKCs. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the mean ΔCt value for miR-221 in OKCs was 11.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91, which was significantly lower than 12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 in DCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). According to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the mean ΔCt value of miR-214 was 10.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 in OKCs and 12.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 in DCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that miR-375 had a mean ΔCt of 8.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 in OKCs versus 11.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 in DCs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression Differences of \u003cem\u003emiR-221\u003c/em\u003e between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean ΔCT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStandard deviation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003elowest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ehighest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOKC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.2691\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.90787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.9523\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.65736\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression Differences of \u003cem\u003emiR-214\u003c/em\u003e between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean ΔCT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStandard deviation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003elowest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ehighest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOKC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.0978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.60916\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.5091\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.45686\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression Differences of \u003cem\u003emiR-375\u003c/em\u003e between odontogenic keratocyst (OKC) and dentigerous cyst (DC) groups based on ΔCt values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean ΔCT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStandard deviation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003elowest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ehighest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOKC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.1803\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.63364\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11.0542\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.86091\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThese ΔCt differences were further translated into relative expression values and fold changes using the 2^(-ΔΔCt) method. As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the fold change of miR-221, miR-214, and miR-375 in OKC tissues was markedly higher than in DC tissues. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e graphically illustrates the differences in ΔCt values between the two groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe calculated relative expression levels and fold changes (Odontogenic keratocyst vs dentigerous cyst).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRelative Expression in OKC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRelative Expression in DC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFold Change (OKC /DC)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.000405\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.000126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-214\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.000911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.000171\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emiR-375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.003448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.000472\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe investigated the diagnostic potential of miR-221, miR-214, and miR-375 expression in OKCs and DCs, due to aggressive growth and higher recurrence rates of OKCs compared with DCs. Our results demonstrate that the expression levels of all three miRNAs in OKCs are significantly elevated in OKC samples relative to DCs. This differential expression suggests that these miRNAs may serve as valuable molecular markers for distinguishing between these two common types of odontogenic cysts. Furthermore, upregulation of certain miRNAs in OKCs may reflect underlying molecular pathways associated with the more proliferative and invasive nature of this lesion. To the best of our knowledge, this is the first study to investigate the expression of these specific miRNAs in odontogenic cysts, highlighting novelty and potential contribution to the field.\u003c/p\u003e\u003cp\u003eEpithelial odontogenic cysts and tumors originate from the odontogenic epithelium and the surface epithelial lining of the oral mucosa. The difference in the proliferation of epithelial cells has an important role in the pathogenesis of these lesions (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The behavior of a lesion is generally associated with its growth potential, which can be assessed by measuring cellular proliferative activity. This growth potential and its correlation with clinical aggressiveness and recurrence can determine the lesion's nature. Experimental studies indicate that the extent of cellular proliferation within a tumor can serve as an indicator of its biological aggression (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Studies have shown that OKCs and DCs can be potential premalignant lesions, and some miRNAs may play a role in this transformation. The change in the expression of these miRNAs is necessary for the initial phases of cancer development. It should be considered evidence of the existence of the potential for malignant transformation of a lesion. Several studies have reported that most of the identified miRNA genes are located within cancer-associated genomic regions, often referred to as fragile sites. As such, identifying these miRNAs may aid in differential diagnosis and assessing malignant potential (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Understanding the role of miRNA in cancer biology is challenging because of its biphasic nature, where the same miRNA can function as an oncomir in one cancer type and as a tumor suppressor in another. For example, miRNA-29 acts as an oncogene in breast cancer but serves as a tumor suppressor in lung cancer. This dual role can be attributed to miRNAs targeting different messenger RNAs (mRNAs) in various tissues, often through diverse pathways (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAccording to past studies, miR-221, miR-214, and miR-375 are not only heavily dysregulated, but also their expression levels in cancer tissues were completely variable (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Recent research has used miRNAs or specific miRNA ratios as prognostic markers in head and neck squamous cell carcinoma. For example, the ratio of miR-221 to miR-375 was highly specific (0.93) and sensitive (0.92) for distinguishing normal from malignant tissues (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMiRNA-221 is part of the miRNA-221/222 cluster encoded in chromosome Xp11.3 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and it has dual roles in cancer as an oncomiR or tumor suppressor (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Overexpression of miR-221 has been linked to increased proliferation in glioblastoma (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), gastric cancer (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), renal cell carcinoma (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), prostate cancer (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and oral squamous cell carcinoma (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, Xie et al. reported that miR-221 inhibits pancreatic cancer cell proliferation through the JAK-STAT3 pathway (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). It has also been shown that miRNA-222 regulates invasion in tongue squamous cell carcinoma by targeting specific enzymes (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Changes in miRNA expression impact signaling pathways and can lead to carcinogenesis and aggressive behavior (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMiR-214 is located on chromosome 1q24.3 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and it was initially identified for its ability to induce apoptosis in HeLa cells by inhibiting the anti-apoptotic protein Bcl2l2 and enhancing pro-apoptotic factors like Bax and caspases (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Additionally, it targets oncogenic mRNAs, including CD44 and CDK1, and leads to reduced proliferation and migration in various cancers (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). MiR-214 exhibits variable expression across cancer types and functions as both a tumor suppressor and an oncogenic miRNA. In Oral Squamous Cell Carcinoma (OSCC), it induces malignancy through the MAPK/ERK pathway and correlates with poor prognosis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), while its inhibition enhances apoptosis (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). A miR-based prognostic model including miR-214-3p appeared to be effective for the detection of early-stage OSCC (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMiR-375 is located on chromosome 2 and plays tissue-specific roles as both a tumor suppressor and an onco-miRNA (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). It suppresses malignancy in nasopharyngeal carcinoma (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) but promotes breast cancer progression by targeting HOXA5 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In oral potentially malignant disorders (OPMD), salivary miR-375 levels are lower in patients with dysplasia, which indicates its potential as a sensitive marker (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In OSCC, downregulation of miR-375 correlates with poor prognosis, reduced survival, and lymph node metastasis (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Elevated miR-375 levels inhibit proliferation and invasion by targeting p53 and SLC7A11, enhancing apoptosis and radiosensitivity (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on the findings of this study and considering that specific miRNAs exhibit high tissue and organ specificity, it seems that assessing the expression of particular miRNAs could be valuable for predicting the clinical behavior of odontogenic cysts. Currently, techniques such as qRT-PCR and microarray are used for miRNA profiling, which are non-invasive, cost-effective, and reliable (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). However, several challenges remain when using miRNAs, such as the complex nature of miRNA-mRNA interactions. A single miRNA can target multiple mRNAs and vice versa. Therefore, standardizing or normalizing miRNA levels in experimental studies of these interactions requires extensive research, which can be challenging. Fortunately, bioinformatics tools and algorithms are available to predict and assess miRNA-mRNA interactions based on sequence data (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Studies from various countries have reported variations in microRNA expression in oral cancer patients, raising concerns about the reliability of miRNAs as biomarkers for cancer. For example, miR-21 levels were higher in plasma from the Japanese and Danish cohorts, but lower in the Turkish cohort and unchanged in Chinese patients (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Consequently, further studies are necessary to provide a clearer explanation. Another limitation of this study was the use of FFPE tissue to extract miRNAs. Since tissue lysis can alter miRNA concentrations, the processing procedure may affect the outcome (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). This emphasizes the need for additional investigations utilizing fresh tissue samples.\u003c/p\u003e\u003cp\u003eThe differential expression of miR-221, miR-214, and miR-375 suggests their potential utility as molecular diagnostic tools. Their application in clinical diagnostics could facilitate early detection and management of odontogenic cysts with aggressive behavior, reducing recurrence and unnecessary extensive surgical interventions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, a significant difference in the expression levels of miR-221, miR-214, and miR-375 was observed between OKCs and DCs. The significant downregulation of these miRNAs in DCs compared with OKCs indicates their potential as diagnostic biomarkers. Therefore, monitoring the expression levels of miRNAs could be used as a valuable tool for the diagnosis and prognosis of odontogenic cysts. These findings provide insights into the molecular differences between OKCs and DCs and facilitate a deeper understanding and evaluation of their clinical behavior.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors confirm that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRobinson RA. Diagnosing the most common odontogenic cystic and osseous lesions of the jaws for the practicing pathologist. Mod Pathol. 2017;30(s1):S96-s103.\u003c/li\u003e\n \u003cli\u003eAli A, Asif M, Ahmad B, Jamal S, Ali I, Khadim MT. Stromal Expression of CD10 by Immunohistochemistry in Odontogenic Keratocyst (OKC), Dentigerous and Radicular Cysts and Its Correlation with Local Recurrence and Aggressive Behaviour. Asian Pac J Cancer Prev. 2019;20(1):249-53.\u003c/li\u003e\n \u003cli\u003eBatra P, Batra SG, Ahuja D, Batra R. Management and Rehabilitation of Dentigerous Cyst With 10-Year Follow-Up: A Case Report. Cureus. 2024;16(8):e67867.\u003c/li\u003e\n \u003cli\u003eA\u0026ccedil;ikg\u0026ouml;z A, Uzun-Bulut E, \u0026Ouml;zden B, G\u0026uuml;nd\u0026uuml;z K. Prevalence and distribution of odontogenic and nonodontogenic cysts in a Turkish population. Med Oral Patol Oral Cir Bucal. 2012;17(1):e108-15.\u003c/li\u003e\n \u003cli\u003eContar CM, Thom\u0026eacute; CA, Pompermayer A, Sarot JR, Vinagre RO, Machado M. Marsupialization of dentigerous cyst: report of a case. 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Cancer Biother Radiopharm. 2020;35(8):615-25.\u003c/li\u003e\n \u003cli\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.\u003c/li\u003e\n \u003cli\u003eDiniz MG, Gomes CC, de Castro WH, Guimar\u0026atilde;es AL, De Paula AM, Amm H, et al. miR-15a/16-1 influences BCL2 expression in keratocystic odontogenic tumors. Cell Oncol (Dordr). 2012;35(4):285-91.\u003c/li\u003e\n \u003cli\u003eModi TG, Chalishazar M, Kumar M. Expression of Ki-67 in odontogenic cysts: A comparative study between odontogenic keratocysts, radicular cysts and dentigerous cysts. J Oral Maxillofac Pathol. 2018;22(1):146.\u003c/li\u003e\n \u003cli\u003eWang X, Guo B, Li Q, Peng J, Yang Z, Wang A, et al. miR-214 targets ATF4 to inhibit bone formation. Nat Med. 2013;19(1):93-100.\u003c/li\u003e\n \u003cli\u003eFidele NB, Yueyu Z, Zhao Y, Tianfu W, Liu J, Sun Y, et al. Recurrence of odontogenic keratocysts and possible prognostic factors: Review of 455 patients. Med Oral Patol Oral Cir Bucal. 2019;24(4):e491-e501.\u003c/li\u003e\n \u003cli\u003eDar GM, Agarwal S, Kumar A, Nimisha, Apurva, Sharma AK, et al. A non-invasive miRNA-based approach in early diagnosis and therapeutics of oral cancer. Crit Rev Oncol Hematol. 2022;180:103850.\u003c/li\u003e\n \u003cli\u003eReddy KB. MicroRNA (miRNA) in cancer. Cancer Cell International. 2015;15(1):38.\u003c/li\u003e\n \u003cli\u003eLakhia R, Yheskel M, Flaten A, Ramalingam H, Aboudehen K, Ferr\u0026egrave; S, et al. Interstitial microRNA miR-214 attenuates inflammation and polycystic kidney disease progression. JCI Insight. 2020;5(7).\u003c/li\u003e\n \u003cli\u003eBasuony S, Hamed RS. Anti-Micro RNA-221 a Promising Genetic Therapy of Oral Squamous Cell Carcinoma (SCC-25). Braz Dent J. 2020;31(6):634-9.\u003c/li\u003e\n \u003cli\u003eAvissar M, Christensen BC, Kelsey KT, Marsit CJ. MicroRNA expression ratio is predictive of head and neck squamous cell carcinoma. Clin Cancer Res. 2009;15(8):2850-5.\u003c/li\u003e\n \u003cli\u003eSukmana BI, Al-Hawary SIS, Abosaooda M, Adile M, Gupta R, Saleh EAM, et al. A thorough and current study of miR-214-related targets in cancer. Pathol Res Pract. 2023;249:154770.\u003c/li\u003e\n \u003cli\u003eWang F, Liu M, Li X, Tang H. MiR-214 reduces cell survival and enhances cisplatin-induced cytotoxicity via down-regulation of Bcl2l2 in cervical cancer cells. FEBS Lett. 2013;587(5):488-95.\u003c/li\u003e\n \u003cli\u003eSharma T, Hamilton R, Mandal CC. miR-214: a potential biomarker and therapeutic for different cancers. Future Oncol. 2015;11(2):349-63.\u003c/li\u003e\n \u003cli\u003eLiu C, Kelnar K, Liu B, Chen X, Calhoun-Davis T, Li H, et al. The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med. 2011;17(2):211-5.\u003c/li\u003e\n \u003cli\u003eDeng N, Xu J. Expression of miR-214 and RASSF5 in oral cancer patients and their effects on apoptosis of oral cancer cells. Int J Clin Exp Med. 2019;12(9):11579-86.\u003c/li\u003e\n \u003cli\u003eBaroukh NN, Van Obberghen E. Function of microRNA-375 and microRNA-124a in pancreas and brain. Febs j. 2009;276(22):6509-21.\u003c/li\u003e\n \u003cli\u003eJia-Yuan X, Wei S, Fang-Fang L, Zhi-Jian D, Long-He C, Sen L. miR-375 Inhibits the Proliferation and Invasion of Nasopharyngeal Carcinoma Cells by Suppressing PDK1. Biomed Res Int. 2020;2020:9704245.\u003c/li\u003e\n \u003cli\u003ede Souza Rocha Simonini P, Breiling A, Gupta N, Malekpour M, Youns M, Omranipour R, et al. Epigenetically deregulated microRNA-375 is involved in a positive feedback loop with estrogen receptor alpha in breast cancer cells. Cancer Res. 2010;70(22):9175-84.\u003c/li\u003e\n \u003cli\u003eMoorthy RK, Srinivasan C, Kannan M, Arockiam AJV. Deregulation of miR-375 Inhibits HOXA5 and Promotes Migration, Invasion, and Cell Proliferation in Breast Cancer. Appl Biochem Biotechnol. 2023;195(7):4503-23.\u003c/li\u003e\n \u003cli\u003eTu HF, Lin LH, Chang KW, Cheng HW, Liu CJ. Exploiting salivary miR-375 as a clinical biomarker of oral potentially malignant disorder. J Dent Sci. 2022;17(2):659-65.\u003c/li\u003e\n \u003cli\u003eNarasimhan S, Narasimhan M, Shetty SR, Rajan ST, Al Kawas S, Subramani VN. Analyzing the Expression of MicroRNA-375 and its Target Gene p53 in Oral Squamous Cell Carcinoma and its Implication in Oral Carcinogenesis. Biomedical and Pharmacology Journal. 2023;16(4):2051-60.\u003c/li\u003e\n \u003cli\u003eWu Y, Sun X, Song B, Qiu X, Zhao J. MiR-375/SLC7A11 axis regulates oral squamous cell carcinoma proliferation and invasion. Cancer Med. 2017;6(7):1686-97.\u003c/li\u003e\n \u003cli\u003eHayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med. 2014;20(8):460-9.\u003c/li\u003e\n \u003cli\u003eMazumder S, Datta S, Ray JG, Chaudhuri K, Chatterjee R. Liquid biopsy: miRNA as a potential biomarker in oral cancer. Cancer Epidemiol. 2019;58:137-45.\u003c/li\u003e\n \u003cli\u003eKirschner MB, Edelman JJ, Kao SC, Vallely MP, van Zandwijk N, Reid G. The Impact of Hemolysis on Cell-Free microRNA Biomarkers. Front Genet. 2013;4:94.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Odontogenic keratocyst, Dentigerous cyst, MicroRNA, miR-221, miR-214, miR-375","lastPublishedDoi":"10.21203/rs.3.rs-7298969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7298969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: The early diagnosis of odontogenic cysts is vital to prevent future complications and choosing an effective treatment plan. Dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) are two common developmental cysts that affect the jaw. OKCs show locally aggressive behavior and a higher recurrence rate compared with DCs, emphasizing the need for reliable molecular diagnostic markers. MicroRNAs (miRNAs) are abnormally expressed in tumors and lesions with aggressive behavior and can be used as diagnostic biomarkers. This study investigates the diagnostic potential of miR-221, miR-214, and miR-375 expression in OKCs and DCs, due to the invasive nature of OKCs compared with DCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e: This study analyzed 36 paraffin-embedded odontogenic cyst tissue samples (23 OKC and 13 DC) collected from the oral pathology archive of Islamic Azad University. Total RNA, including miRNAs, was extracted using TRIzol-based protocols with RNase-free conditions. RNA purity and integrity were assessed via spectrophotometry and agarose gel electrophoresis. Reverse transcription was performed using the M-MLV RNase H kit. Specific primers for miR-221, miR-214, and miR-375 were designed and verified. Quantitative real-time PCR was conducted using SYBR Green, and delta cycle threshold (ΔCt) values were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: miR-221, miR-214, and miR-375 showed significantly lower ΔCt values in OKCs compared with DCs (p \u0026lt; 0.001). The mean ΔCt values for miR-221, miR-214, and miR-375 in OKCs were 11.27, 10.10, and 8.18, respectively, compared with 12.95, 12.51, and 11.05 in DCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The ΔCt values for miR-221, miR-214, and miR-375s were significantly lower in OKC samples, indicating higher expression levels compared with DCs.The significant upregulation of these miRNAs in OKCs compared with DCs suggests that these miRNAs have potential as diagnostic biomarkers.\u003c/p\u003e","manuscriptTitle":"Diagnostic Potential of miR-221, miR-214, and miR-375 Expression in Odontogenic Keratocysts Versus Dentigerous Cysts: An In Vitro Comparison","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 12:33:44","doi":"10.21203/rs.3.rs-7298969/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-15T08:09:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T04:30:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T20:59:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-12T22:14:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T03:24:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90795115848842921902657537074753339195","date":"2025-08-27T07:29:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255818408598404087661173589589232476276","date":"2025-08-24T21:40:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130938137633770455128334866459808583361","date":"2025-08-24T08:38:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24557675596454218907239083048265181153","date":"2025-08-24T06:50:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18058557573980593918282373566258345616","date":"2025-08-23T08:35:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-22T05:59:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-13T09:15:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T06:50:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-13T06:49:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-08-05T09:34:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d932fb48-5c4c-4b33-827e-184e589aa31e","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:06:46+00:00","versionOfRecord":{"articleIdentity":"rs-7298969","link":"https://doi.org/10.1186/s12903-025-07289-0","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2025-12-12 15:58:03","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-09-01 12:33:44","video":"","vorDoi":"10.1186/s12903-025-07289-0","vorDoiUrl":"https://doi.org/10.1186/s12903-025-07289-0","workflowStages":[]},"version":"v1","identity":"rs-7298969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7298969","identity":"rs-7298969","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}