Combined Effects of 5-Azacytidine and Oleuropein on miR-149-3p, miR-375, miR-574-5p Expression and Apoptosis in HL-60 and THP-1 Cell Lines

Combined Effects of 5-Azacytidine and Oleuropein on miR-149-3p, miR-375, miR-574-5p Expression and Apoptosis in HL-60 and THP-1 Cell Lines | 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 Combined Effects of 5-Azacytidine and Oleuropein on miR-149-3p, miR-375, miR-574-5p Expression and Apoptosis in HL-60 and THP-1 Cell Lines Shohre Karimi Kelaye, Bahareh Kazemi, Fatemeh Najafi, Zahra Foruzandeh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7091096/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by the rapid expansion of immature myeloid cells and poor clinical outcomes. Despite conventional treatments, including chemotherapy and hematopoietic stem cell transplantation, relapse and resistance remain significant challenges. Epigenetic alterations, particularly dysregulated DNA methylation and microRNA (miRNA) expression, play a crucial role in AML pathogenesis. Objective This study aimed to evaluate the synergistic effects of azacitidine, a DNA methyltransferase inhibitor, and oleuropein, a natural polyphenol with anticancer properties, on AML cell lines (THP-1 and HL-60). Methods AML cells were treated with azacitidine, oleuropein, and their combination. Cell proliferation was assessed using MTT assays, apoptosis was analyzed via flow cytometry (Annexin V-FITC/PI staining), and miRNA expression levels were quantified using real-time PCR. Results Both azacitidine and oleuropein reduced cell viability and induced apoptosis in a dose- and time-dependent manner. Notably, the combination treatment significantly enhanced apoptosis, with a 2.5-fold increase in Annexin V-positive HL-60 cells at 72 hours. Furthermore, the treatment modulated miRNA expression, upregulating miR-149-3p and miR-375 while downregulating miR-574-5p. Conclusion The synergistic effects of oleuropein and azacitidine suggest a potential therapeutic strategy for AML by targeting epigenetic mechanisms and miRNA pathways. Further in vivo studies and clinical trials are necessary to validate these findings and optimize treatment protocols. Acute myeloid leukemia azacitidine oleuropein microRNA mir-149-3p mir-375 mir-574-5p Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Acute myeloid leukemia (AML) is a heterogeneous malignancy originating from hematopoietic stem cell progenitors, characterized by the clonal expansion of immature myeloid cells. Although AML accounts for approximately one-third of all leukemia cases, its aggressive clinical course, high relapse rates, and resistance to conventional therapies (such as intensive chemotherapy and hematopoietic stem cell transplantation) remain significant challenges[1]. Recent research has increasingly implicated epigenetic dysregulation—including aberrant DNA methylation, histone modifications, and miRNA imbalances—in the pathogenesis of AML. These epigenetic alterations not only drive leukemogenesis but also contribute to therapeutic resistance and disease relapse[2]. Azacitidine, a hypomethylating agent, has been employed to reverse DNA hypermethylation and reactivate silenced tumor suppressor genes. However, its clinical efficacy is frequently limited by the persistence of leukemic stem cells and the emergence of resistance mechanisms. This limitation has prompted researchers to explore combination strategies that target complementary molecular pathways. In this context, natural compounds with diverse bioactive properties have garnered significant attention[3]. Oleuropein, a polyphenol richly present in olive leaves and fruits, possesses strong antioxidant, anti-inflammatory, and anticancer properties. Preclinical research has shown that oleuropein can suppress angiogenesis, decrease cell proliferation, and trigger apoptosis by regulating critical signaling pathways, including NF-κB, PI3K/Akt, and Wnt/β-catenin[4]. Moreover, recent studies suggest that oleuropein can modulate the expression of microRNAs (miRNAs), which are small non-coding RNAs playing vital roles in the post-transcriptional regulation of gene expression[5]. The dysregulation of specific miRNAs has been linked to both the initiation and progression of AML, rendering miRNA modulation an attractive therapeutic target[6]. In particular, miR-149-3p, miR-375, and miR-574-5p have garnered attention due to their involvement in cell cycle regulation and apoptosis. The dysregulation of these miRNAs has been linked to both the initiation and progression of AML, making them attractive therapeutic targets[7]. By modulating the expression of these miRNAs, novel treatment strategies may overcome some of the limitations associated with conventional therapies and contribute to improved outcomes in AML[8]. Given these insights, the present study aims to evaluate the antiproliferative and pro-apoptotic effects of azacitidine and oleuropein—administered alone and in combination—on AML cell lines (THP-1 and HL-60). Moreover, we seek to elucidate the impact of these agents on the expression of key miRNAs (miR-149-3p, miR-375, and miR-574-5p) that are implicated in cell cycle regulation and apoptosis. By addressing the existing gaps in our understanding of AML epigenetics, this study not only reinforces the rationale for combination therapy but also proposes advanced molecular analyses (such as RNA-Seq and proteomics) as future steps to comprehensively profile the downstream effects of the treatment. 2. Materials and Methods 2.1. Cell Lines and Culture Conditions: Human promyeloblast leukemic cells (HL-60) and human monocytic cells (THP-1) were obtained from the Pasteur Institute of Iran. Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.3 mg/ml L-glutamine, and antibiotics (100 IU penicillin and 100 mg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO₂. 2.2. Cell Viability Analysis (MTT Assay): To evaluate the antiproliferative effects of oleuropein and azacitidine, 1×10⁴ cells were seeded in 96-well plates and treated with a range of concentrations of oleuropein (0–220 µM) and azacitidine (0–320 µM) for 24, 48, and 72 hours. After treatment, 0.1 mg/ml MTT was added and cells were incubated for an additional 4 hours at 37°C. Formazan crystals were dissolved in 100 µl of a solution containing 0.01 M SDS in 0.01 M HCl, and absorbance was measured at 570 nm. IC50 values were determined from cell proliferation plots. 2.3. Assessment of Apoptosis by Annexin V-FITC/PI Staining: Cells were divided into four groups: untreated control, oleuropein-treated, azacitidine-treated, and combination treatment. Based on IC50 values, cells were treated for 24, 48, and 72 hours. After treatment, cells were collected, washed twice with cold PBS, and resuspended in 200 µl of binding buffer. Then, 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) were added, and the samples were incubated for 30 minutes at room temperature in the dark. Samples were analyzed by flow cytometry using FlowJo v10 software. Data are presented as mean ± SEM, and statistical significance was determined using two-way ANOVA (p < 0.05). 2.4. RNA Isolation and cDNA Synthesis: Total RNA was isolated using TRIzol reagent (Qiagen, Germany) following the manufacturer’s protocol. RNA purity and concentration were assessed with a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized from total RNA using a stem-loop primer-based cDNA synthesis kit (Yekta Tajhiz) specific for miR-149-3p, miR-375, miR-574-5p, with U6 serving as the internal control. The reaction was performed with sequential incubations at 16°C (30 minutes), 42°C (30 minutes), and 72°C (5 minutes) for enzyme inactivation. 2.5. Quantitative Real-Time PCR (qRT-PCR) Analysis: cDNA was amplified using SYBR Green master mix in a Rotor-Gene Q thermocycler. Each reaction contained SYBR Green, ddH₂O, specific forward primers, a universal reverse primer, and 0.5 µl of cDNA. Expression levels of miR-149-3p, miR-375, and miR-574-5p were normalized to U6, and fold changes were calculated using the 2^–ΔΔCt method. 2.6. Statistical Analysis: Data are expressed as mean ± SEM of at least three independent experiments. Statistical differences between groups were evaluated using one-way ANOVA followed by Dunnett's post hoc test (p < 0.05 was considered statistically significant) using GraphPad Prism v9.1.1. 3. Results 3.1. Effects on Cell Viability: MTT assay results indicated that both azacitidine and oleuropein reduced cell viability in THP-1 and HL-60 cells in a dose- and time-dependent manner. In THP-1 cells, the IC50 values for azacitidine were 36.56 µM (24 h), 30.41 µM (48 h), and 23.62 µM (72 h), while oleuropein induced approximately 50% cell death at concentrations of 162 µM, 146.8 µM, and 134.6 µM at 24, 48, and 72 hours, respectively. In HL-60 cells, the IC50 values for azacitidine were 15.23 µM (24 h), 7.368 µM (48 h), and 5.906 µM (72 h), with oleuropein achieving 50% cell death at 144.5 µM, 131.8 µM, and 119.2 µM at 24, 48, and 72 hours, respectively. 3.2. Apoptosis Analysis: Flow cytometry using Annexin V-FITC/PI staining revealed that both agents increased apoptosis in AML cell lines. Notably, the combination treatment produced a synergistic effect, leading to a significant increase in the percentage of apoptotic cells. For example, in HL-60 cells, a 2.5-fold increase in Annexin V-positive cells was observed at 72 hours compared to untreated controls. 3.3. Modulation of miRNA Expression: qRT-PCR analysis demonstrated that treatment with azacitidine and oleuropein, both individually and in combination, significantly modulated miRNA expression in AML cell lines. Specifically, miR-149-3p and miR-375 levels were upregulated, while miR-574-5p was downregulated in a dose- and time-dependent manner. The combination treatment resulted in the most pronounced changes, particularly after 72 hours. 4. Discussion AML remains a formidable clinical challenge due to its aggressive nature and resistance to conventional therapies. The persistence of epigenetic abnormalities and aberrant miRNA expression contributes to the disease's pathogenesis and treatment failure[6]. Azacitidine, although effective as a hypomethylating agent, is hampered by limited efficacy and high relapse rates[9]. Our study proposes that combining azacitidine with oleuropein may overcome some of these limitations by targeting complementary pathways. Recent studies have highlighted the significant role of epigenetic changes in the pathogenesis of AML and its resistance to standard therapies. Key epigenetic mechanisms include alterations in DNA methylation patterns, abnormal histone modifications, and the dysregulation of non-coding RNAs, such as microRNAs (miRNAs)[3]. Azacitidine, a DNA methylation inhibitor, has shown promise by reactivating silenced tumor suppressor genes. However, its clinical application is limited due to significant side effects and the development of resistance in the advanced stages of the disease. Consequently, researchers are increasingly exploring combination therapies that can enhance the effectiveness of azacitidine while minimizing its drawbacks[10]. Natural compounds with well-established anticancer properties are gaining attention as potential adjuncts to conventional therapies. Oleuropein, a polyphenol present in olive leaves, exhibits diverse biological activities, encompassing anti-inflammatory, antioxidant, and anticancer effects. Preclinical studies have shown that oleuropein can promote apoptosis, block angiogenesis, and regulate critical signaling pathways, including NF-κB, PI3K/Akt, and Wnt/β-catenin[11]. Specifically, oleuropein has been shown to trigger apoptosis by enhancing the levels of caspase-3, 8, and 9, while inhibiting the PI3K-Akt pathway in the HepG2 human liver cancer cell line[12]. Research by Sophia R. Fagan et al. also found that oleuropein inhibits the growth and survival of human leukemia K562 cells by downregulating Peroxiredoxin-1 (Prdx1), a critical antioxidant in blood cells. These results highlight oleuropein as a potential adjunct to conventional cancer therapies, with the ability to enhance treatment efficacy, minimize side effects, and improve overall therapeutic outcomes[13]. ABTIN et al. reported that 600 µM of oleuropein was required for 50% suppression of breast cancer MCF-7 cells[14], while 170 µM significantly decreased cell viability in leukemia HL-60 cells. Furthermore, the IC50 of azacitidine for THP-1 and HL-60 cells has been determined to be 0.5 µM and 1 µM, respectively[9]. In our study, both azacitidine and oleuropein inhibited the proliferation of THP-1 and HL-60 cells and induced apoptosis. The cytotoxic properties of these agents were assessed using the MTT assay, which revealed time-dependent effects (Table 3,4,5,6). Flow cytometry analysis, using Annexin V and propidium iodide (PI) staining, demonstrated that both oleuropein and azacitidine induced apoptotic cell death, with the highest levels of apoptosis observed 72 hours after treatment with both agents (Fig. 9,12). A key feature of oleuropein’s anticancer activity is its capacity to modulate miRNA expression. MiRNAs are essential regulators of various cellular processes, such as cell proliferation, differentiation, apoptosis, and metastasis. For instance, in breast cancer cells (MCF-7), oleuropein has been shown to increase apoptosis by decreasing the expression of miR-21 and miR-155[14]. In AML, dysregulated miRNAs are commonly observed, with some acting as oncogenes and others as tumor suppressors. Given this context, investigating the impact of oleuropein on miRNA expression in AML could provide valuable insights into its potential as a therapeutic agent[6]. This study explores the combined effects of oleuropein and azacitidine in AML treatment, specifically focusing on whether oleuropein can potentiate the therapeutic effects of azacitidine while minimizing its adverse effects. By examining the synergistic potential of these two compounds, our goal is to advance the development of more effective and patient-centered approaches for AML treatment. Further research is required to fully elucidate the molecular mechanisms underlying this combination and to optimize its clinical application. In this study, we examined the effects of oleuropein, both alone and in combination with azacitidine, on apoptosis and the expression of three miRNAs (miR-149-3p, miR-375, and miR-574-5p) in AML cell lines (THP-1 and HL-60). Our findings reveal critical insights into the regulatory roles of these miRNAs in AML pathogenesis and their potential therapeutic implications. Our results indicate that miR-149-3p expression was significantly increased following treatment with azacitidine and oleuropein, either alone or in combination. This aligns with previous findings by MINGJUN LU et al, which reported that azacitidine upregulates miR-149-3p expression[15]. While some studies have suggested an oncogenic role for miR-149-3p in T-cell ALL[16], our findings support its tumor-suppressive function in AML, consistent with its previously reported ability to inhibit the Akt1 signaling pathway and suppress bladder cancer proliferation[17]. Notably, our study extends these findings by demonstrating that oleuropein also contributes to miR-149-3p upregulation, suggesting its potential as a therapeutic agent in AML. Similarly, our results showed a significant increase in miR-375 expression in HL-60 and THP-1 cells treated with azacitidine and oleuropein. This aligns with prior research indicating that miR-375 functions as a tumor suppressor in multiple cancers, including AML. While miR-375 has been identified as an oncogene in some cancers, such as prostate cancer and SCLC[18], our study supports its tumor-suppressive role in AML. We further demonstrated that oleuropein enhances miR-375 expression, and when combined with azacitidine, the upregulation effect was more pronounced. These findings highlight the potential of targeting the miR-375-HOXB3-CDCA3/DNMT3B regulatory axis for AML therapy[8]. Conversely, miR-574-5p was significantly downregulated following treatment with azacitidine and oleuropein, supporting its role as a potential oncogenic miRNA in AML. Previous studies have demonstrated that miR-574-5p promotes proliferation and migration in NSCLC[19], colon cancer, and nasopharyngeal carcinoma via pathways such as β-catenin/Wnt[20]. While its role in leukemia has been less explored, our results suggest that suppressing miR-574-5p expression may be a viable therapeutic strategy. The reduction in miR-574-5p levels following oleuropein and azacitidine treatment provides new insights into its regulatory role in AML and highlights the potential of combination therapy for targeted miRNA modulation. Overall, our findings underscore the importance of miRNA regulation in AML and suggest that azacitidine and oleuropein, either individually or in combination, may serve as effective modulators of miRNA expression. Future research should aim to clarify the specific molecular mechanisms driving these effects and assess their therapeutic potential in clinical environments. 5. Limitations and Future Directions While our in vitro study provides compelling evidence for the synergistic effects of oleuropein and azacitidine in AML, several limitations should be addressed in future work: In Vivo Validation: Future studies should evaluate the combination therapy in animal models to assess pharmacokinetics, biodistribution, and therapeutic efficacy. Mechanistic Insights: Detailed investigation of the molecular pathways affected by the combination treatment—such as its impact on DNMT activity, histone modifications, and broader gene expression patterns using RNA-Seq—is warranted. Clinical Translation: Dose optimization studies and clinical trials are needed to determine the safety, tolerability, and long-term efficacy of this combination, particularly in elderly patients and those with comorbidities. 6. Conclusion Our study demonstrates that the combination of oleuropein and azacitidine significantly enhances therapeutic outcomes in AML by inducing apoptosis and modulating key miRNAs (upregulating miR-149-3p and miR-375, and downregulating miR-574-5p). These findings suggest that combination therapy not only potentiates the effects of azacitidine but may also provide a less toxic alternative to high-dose chemotherapy. This research represents one of the first systematic investigations into the miRNA-modulatory effects of oleuropein in AML, paving the way for further in vivo studies and clinical trials to optimize this promising therapeutic strategy. Declarations Ethics approval and consent to participate: This study was approved by the Ethics Committee of Tabriz University of Medical Sciences, Tabriz, Iran Consent for publication: Not Applicable Availability of data and materials: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Competing Interests: The authors declare no conflict of interest. Author's Contribution: S. Karimi Kelaye conceived and designed the study, performed the majority of the experimental work, analyzed the data, and drafted the initial manuscript, B. Kazemi participated in data collection and performed statistical analyses, Z. Foruzandeh and F.Najafi were responsible for additional data acquisition, S Solali supervised the study, provided essential guidance, coordinated research activities, and critically revised the final manuscript. All authors have read and approved the final manuscript. Funding : This research was financially supported by the Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Acknowledgement : We gratefully acknowledge the staff of the Division of Hematology and Blood Banking, Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, for their valuable support. References Fennell KA, Bell CC, Dawson MA. Epigenetic therapies in acute myeloid leukemia: where to from here? [Internet]. 2019. Available from: https://ashpublications.org/blood/article-pdf/134/22/1891/1543146/bloodbld2019003262.pdf Issah MA, Wu D, Zhang F, Zheng W, Liu Y, Fu H, et al. Epigenetic modifications in acute myeloid leukemia: The emerging role of circular RNAs (Review). Vol. 59, International Journal of Oncology. Spandidos Publications; 2021. Karimi Kelaye S, Najafi F, Kazemi B, Foruzandeh Z, Seif F, Solali S, et al. The contributing factors of resistance or sensitivity to epigenetic drugs in the treatment of AML. Vol. 24, Clinical and Translational Oncology. Springer Science and Business Media Deutschland GmbH; 2022. p. 1250–61. Rishmawi S, Haddad F, Dokmak G, Karaman R. A Comprehensive Review on the Anti-Cancer Effects of Oleuropein. Vol. 12, Life. MDPI; 2022. Mahi H, Yousefi Z, Toufan F, Yarmohammadi M, Jafarisani M, Eskandari N, et al. Oleuropein as An Effective Suppressor of Inflammation and MicroRNA-146a Expression in Patients with Rheumatoid Arthritis. Cell J. 2023;25(7):505–12. Chandraprabha Vineetha R, Anitha Geetha Raj J, Devipriya P, Sreelatha Mahitha M, Hariharan S. MicroRNA-based therapies: Revolutionizing the treatment of acute myeloid leukemia. Vol. 46, International Journal of Laboratory Hematology. John Wiley and Sons Inc; 2024. p. 33–41. Tian Y, Jiang Y, Dong X, Chang Y, Chi J, Chen X. miR-149-3p suppressed epithelial–mesenchymal transition and tumor development in acute myeloid leukemia. Hematology (United Kingdom). 2021;26(1):840–7. Bi L, Zhou B, Li H, He L, Wang C, Wang Z, et al. A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia. BMC Cancer. 2018 Feb 13;18(1). Xiao X, Xu Q, Sun Y, Lu Z, Li R, Wang X, et al. 5-aza-2′-deoxycytidine promotes migration of acute monocytic leukemia cells via activation of CCL2-CCR2-ERK signaling pathway. Mol Med Rep. 2017 Aug 1;16(2):1417–24. Wang X, Xiao Z, Qin T, Xu Z, Jia Y, Qu S, et al. Combination therapy with venetoclax and azacitidine for the treatment of myelodysplastic syndromes with DDX41 mutations. Hematology (United Kingdom). 2024;29(1). Shamshoum H, Vlavcheski F, Tsiani E. Anticancer effects of oleuropein. Vol. 43, BioFactors. Blackwell Publishing Inc.; 2017. p. 517–28. Yan CM, Chai EQ, Cai HY, Miao GY, Ma W. Oleuropein induces apoptosis via activation of caspases and suppression of phosphatidylinositol 3-kinase/protein kinase B pathway in HepG2 human hepatoma cell line. Mol Med Rep. 2015 Jun 1;11(6):4617–24. Oleuropein Reduces Prdx1 Expression, Cell Proliferation and Viability in K562 Human Leukemia Cells. ARC Journal of Cancer Science. 2019;5(1). Abtin M, Alivand MR, Khaniani MS, Bastami M, Zaeifizadeh M, Derakhshan SM. Simultaneous downregulation of miR-21 and miR-155 through oleuropein for breast cancer prevention and therapy. J Cell Biochem. 2018 Sep 1;119(9):7151–65. Lu M, Xu L, Wang M, Guo T, Luo F, Su N, et al. MiR-149 promotes the myocardial differentiation of mouse bone marrow stem cells by targeting Dab2. Mol Med Rep. 2018 Jun 1;17(6):8502–9. Zhang M, Gao D, Shi Y, Wang Y, Joshi R, Yu Q, et al. miR-149-3p reverses CD8+ T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells. Open Biol. 2019 Oct 31;9(10):190061. Yang D, Du G, Xu A, Xi X, Li D. Expression of miR-149-3p inhibits proliferation, migration, and invasion of bladder cancer by targeting S100A4 [Internet]. Vol. 7, Am J Cancer Res. 2017. Available from: www.ajcr.us/ISSN:2156-6976/ajcr0064090 Yan JW, Lin JS, He XX. The emerging role of miR-375 in cancer. Vol. 135, International Journal of Cancer. Wiley-Liss Inc.; 2014. p. 1011–8. Zhou R, Zhou X, Yin Z, Guo J, Hu T, Jiang S, et al. MicroRNA-574-5p promotes metastasis of non-small cell lung cancer by targeting PTPRU. Sci Rep. 2016 Oct 20;6. Lin Z, Chen M, Wan Y, Lei L, Ruan H. miR-574-5p Targets FOXN3 to Regulate the Invasion of Nasopharyngeal Carcinoma Cells via Wnt/β-Catenin Pathway. Technol Cancer Res Treat. 2020;19. Additional Declarations No competing interests reported. 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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-7091096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485487464,"identity":"04d83385-0a12-418c-b6ac-13dd7a2515a0","order_by":0,"name":"Shohre Karimi Kelaye","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shohre","middleName":"Karimi","lastName":"Kelaye","suffix":""},{"id":485487465,"identity":"1b253139-d526-4007-a1e7-6174b16eb47d","order_by":1,"name":"Bahareh Kazemi","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bahareh","middleName":"","lastName":"Kazemi","suffix":""},{"id":485487466,"identity":"8840548e-6533-49d3-b3e9-486146d42fcd","order_by":2,"name":"Fatemeh Najafi","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Najafi","suffix":""},{"id":485487467,"identity":"f9a85e8f-69e9-4053-9d4b-eb19c375c822","order_by":3,"name":"Zahra Foruzandeh","email":"","orcid":"","institution":"Genetic Research Div. 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Data represent mean ± SD, with IC₅₀ values shown in the graph tables. The comparison includes four groups: untreated control, azacitidine (Aza), oleuropein (Oleu), and their combination (Mix). Significant differences among groups are indicated by p-values.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/c55445092d894fb3db7c246a.png"},{"id":86951141,"identity":"9966caf8-8518-453a-a5f6-fa78a3cef15b","added_by":"auto","created_at":"2025-07-17 14:17:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCell viability of HL-60 cells treated with azacitidine, oleuropein, and their combination at 24, 48, and 72 hours. Data represent mean ± SD, with IC₅₀ values shown in the graph tables. The comparison includes four groups: untreated control, azacitidine (Aza), oleuropein (Oleu), and their combination (Mix). Significant differences among groups are indicated by p-values.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/a39b914d2964ce8221fdd601.png"},{"id":86951944,"identity":"83ad7a33-08d8-48a6-80d3-d96fbea17e40","added_by":"auto","created_at":"2025-07-17 14:25:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFlow cytometric analysis of apoptosis in THP-1 and HL-60 cells after 24, 48, and 72 hours of treatment. Cells were categorized into four groups: untreated, azacitidine (Aza), oleuropein (Oleu), and their combination (Mix). Representative dot plots illustrate each treatment condition, and the bar chart summarizes apoptotic cell percentages in each group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/79e93ff60f7a6453b3a1462b.png"},{"id":86950844,"identity":"b20364c4-1b6c-484f-a0ff-1279adddde82","added_by":"auto","created_at":"2025-07-17 14:09:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative expression levels of miR-149-3p in THP-1 and HL-60 cells at 24, 48, and 72 hours under different treatments (Untreated, Oleu, Aza, and Mix). Expression was analyzed via real-time PCR and presented as fold change relative to the untreated group. A time-dependent increase was observed, with the highest expression in the Mix group at 72 hours. Statistical significance is indicated (p-values)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/f6487b85071d00a0b2a2032b.png"},{"id":86950849,"identity":"a9924bf1-cbd4-437e-b513-790e64b04f6f","added_by":"auto","created_at":"2025-07-17 14:09:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative expression levels of miR-375 in THP-1 and HL-60 cells at 24, 48, and 72 hours under different treatments (Untreated, Oleu, Aza, and Mix). Expression was analyzed via real-time PCR and presented as fold change relative to the untreated group. A time-dependent increase was observed, with the highest expression in the Mix group at 72 hours. Statistical significance is indicated (p-values)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/ac28ea6f8d6728ebb659da5a.png"},{"id":86950848,"identity":"ab428a0e-8426-4dd1-98b3-7cb20e236306","added_by":"auto","created_at":"2025-07-17 14:09:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative expression levels of miR-574-5p in THP-1 and HL-60 cells at 24, 48, and 72 hours under different treatments (Untreated, Oleu, Aza, and Mix). Expression was analyzed via real-time PCR and presented as fold change relative to the untreated group. A time-dependent decrease was observed, with the lowest expression in the Mix group at 72 hours. Statistical significance is indicated (p-values).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/6121f8d49ae417bce4028d7b.png"},{"id":93956749,"identity":"1ed7d1e4-913a-49fc-b348-7906e9edb354","added_by":"auto","created_at":"2025-10-20 16:12:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1108459,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7091096/v1/72eb50b8-a383-4a48-bd5d-52b7791c8a8a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combined Effects of 5-Azacytidine and Oleuropein on miR-149-3p, miR-375, miR-574-5p Expression and Apoptosis in HL-60 and THP-1 Cell Lines","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) is a heterogeneous malignancy originating from hematopoietic stem cell progenitors, characterized by the clonal expansion of immature myeloid cells. Although AML accounts for approximately one-third of all leukemia cases, its aggressive clinical course, high relapse rates, and resistance to conventional therapies (such as intensive chemotherapy and hematopoietic stem cell transplantation) remain significant challenges[1]. Recent research has increasingly implicated epigenetic dysregulation\u0026mdash;including aberrant DNA methylation, histone modifications, and miRNA imbalances\u0026mdash;in the pathogenesis of AML. These epigenetic alterations not only drive leukemogenesis but also contribute to therapeutic resistance and disease relapse[2].\u003c/p\u003e\u003cp\u003eAzacitidine, a hypomethylating agent, has been employed to reverse DNA hypermethylation and reactivate silenced tumor suppressor genes. However, its clinical efficacy is frequently limited by the persistence of leukemic stem cells and the emergence of resistance mechanisms. This limitation has prompted researchers to explore combination strategies that target complementary molecular pathways. In this context, natural compounds with diverse bioactive properties have garnered significant attention[3].\u003c/p\u003e\u003cp\u003eOleuropein, a polyphenol richly present in olive leaves and fruits, possesses strong antioxidant, anti-inflammatory, and anticancer properties. Preclinical research has shown that oleuropein can suppress angiogenesis, decrease cell proliferation, and trigger apoptosis by regulating critical signaling pathways, including NF-κB, PI3K/Akt, and Wnt/β-catenin[4]. Moreover, recent studies suggest that oleuropein can modulate the expression of microRNAs (miRNAs), which are small non-coding RNAs playing vital roles in the post-transcriptional regulation of gene expression[5]. The dysregulation of specific miRNAs has been linked to both the initiation and progression of AML, rendering miRNA modulation an attractive therapeutic target[6].\u003c/p\u003e\u003cp\u003eIn particular, miR-149-3p, miR-375, and miR-574-5p have garnered attention due to their involvement in cell cycle regulation and apoptosis. The dysregulation of these miRNAs has been linked to both the initiation and progression of AML, making them attractive therapeutic targets[7]. By modulating the expression of these miRNAs, novel treatment strategies may overcome some of the limitations associated with conventional therapies and contribute to improved outcomes in AML[8].\u003c/p\u003e\u003cp\u003eGiven these insights, the present study aims to evaluate the antiproliferative and pro-apoptotic effects of azacitidine and oleuropein\u0026mdash;administered alone and in combination\u0026mdash;on AML cell lines (THP-1 and HL-60). Moreover, we seek to elucidate the impact of these agents on the expression of key miRNAs (miR-149-3p, miR-375, and miR-574-5p) that are implicated in cell cycle regulation and apoptosis. By addressing the existing gaps in our understanding of AML epigenetics, this study not only reinforces the rationale for combination therapy but also proposes advanced molecular analyses (such as RNA-Seq and proteomics) as future steps to comprehensively profile the downstream effects of the treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell Lines and Culture Conditions:\u003c/h2\u003e\u003cp\u003eHuman promyeloblast leukemic cells (HL-60) and human monocytic cells (THP-1) were obtained from the Pasteur Institute of Iran. Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.3 mg/ml L-glutamine, and antibiotics (100 IU penicillin and 100 mg/ml streptomycin) at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell Viability Analysis (MTT Assay):\u003c/h2\u003e\u003cp\u003eTo evaluate the antiproliferative effects of oleuropein and azacitidine, 1\u0026times;10⁴ cells were seeded in 96-well plates and treated with a range of concentrations of oleuropein (0\u0026ndash;220 \u0026micro;M) and azacitidine (0\u0026ndash;320 \u0026micro;M) for 24, 48, and 72 hours. After treatment, 0.1 mg/ml MTT was added and cells were incubated for an additional 4 hours at 37\u0026deg;C. Formazan crystals were dissolved in 100 \u0026micro;l of a solution containing 0.01 M SDS in 0.01 M HCl, and absorbance was measured at 570 nm. IC50 values were determined from cell proliferation plots.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Assessment of Apoptosis by Annexin V-FITC/PI Staining:\u003c/h2\u003e\u003cp\u003eCells were divided into four groups: untreated control, oleuropein-treated, azacitidine-treated, and combination treatment. Based on IC50 values, cells were treated for 24, 48, and 72 hours. After treatment, cells were collected, washed twice with cold PBS, and resuspended in 200 \u0026micro;l of binding buffer. Then, 5 \u0026micro;l of Annexin V-FITC and 5 \u0026micro;l of propidium iodide (PI) were added, and the samples were incubated for 30 minutes at room temperature in the dark. Samples were analyzed by flow cytometry using FlowJo v10 software. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and statistical significance was determined using two-way ANOVA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. RNA Isolation and cDNA Synthesis:\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated using TRIzol reagent (Qiagen, Germany) following the manufacturer\u0026rsquo;s protocol. RNA purity and concentration were assessed with a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized from total RNA using a stem-loop primer-based cDNA synthesis kit (Yekta Tajhiz) specific for miR-149-3p, miR-375, miR-574-5p, with U6 serving as the internal control. The reaction was performed with sequential incubations at 16\u0026deg;C (30 minutes), 42\u0026deg;C (30 minutes), and 72\u0026deg;C (5 minutes) for enzyme inactivation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Quantitative Real-Time PCR (qRT-PCR) Analysis:\u003c/h2\u003e\u003cp\u003ecDNA was amplified using SYBR Green master mix in a Rotor-Gene Q thermocycler. Each reaction contained SYBR Green, ddH₂O, specific forward primers, a universal reverse primer, and 0.5 \u0026micro;l of cDNA. Expression levels of miR-149-3p, miR-375, and miR-574-5p were normalized to U6, and fold changes were calculated using the 2^\u0026ndash;ΔΔCt method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Statistical Analysis:\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of at least three independent experiments. Statistical differences between groups were evaluated using one-way ANOVA followed by Dunnett's post hoc test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant) using GraphPad Prism v9.1.1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effects on Cell Viability:\u003c/h2\u003e\u003cp\u003eMTT assay results indicated that both azacitidine and oleuropein reduced cell viability in THP-1 and HL-60 cells in a dose- and time-dependent manner. In THP-1 cells, the IC50 values for azacitidine were 36.56 \u0026micro;M (24 h), 30.41 \u0026micro;M (48 h), and 23.62 \u0026micro;M (72 h), while oleuropein induced approximately 50% cell death at concentrations of 162 \u0026micro;M, 146.8 \u0026micro;M, and 134.6 \u0026micro;M at 24, 48, and 72 hours, respectively. In HL-60 cells, the IC50 values for azacitidine were 15.23 \u0026micro;M (24 h), 7.368 \u0026micro;M (48 h), and 5.906 \u0026micro;M (72 h), with oleuropein achieving 50% cell death at 144.5 \u0026micro;M, 131.8 \u0026micro;M, and 119.2 \u0026micro;M at 24, 48, and 72 hours, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Apoptosis Analysis:\u003c/h2\u003e\u003cp\u003eFlow cytometry using Annexin V-FITC/PI staining revealed that both agents increased apoptosis in AML cell lines. Notably, the combination treatment produced a synergistic effect, leading to a significant increase in the percentage of apoptotic cells. For example, in HL-60 cells, a 2.5-fold increase in Annexin V-positive cells was observed at 72 hours compared to untreated controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Modulation of miRNA Expression:\u003c/h2\u003e\u003cp\u003eqRT-PCR analysis demonstrated that treatment with azacitidine and oleuropein, both individually and in combination, significantly modulated miRNA expression in AML cell lines. Specifically, miR-149-3p and miR-375 levels were upregulated, while miR-574-5p was downregulated in a dose- and time-dependent manner. The combination treatment resulted in the most pronounced changes, particularly after 72 hours.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAML remains a formidable clinical challenge due to its aggressive nature and resistance to conventional therapies. The persistence of epigenetic abnormalities and aberrant miRNA expression contributes to the disease's pathogenesis and treatment failure[6]. Azacitidine, although effective as a hypomethylating agent, is hampered by limited efficacy and high relapse rates[9]. Our study proposes that combining azacitidine with oleuropein may overcome some of these limitations by targeting complementary pathways.\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the significant role of epigenetic changes in the pathogenesis of AML and its resistance to standard therapies. Key epigenetic mechanisms include alterations in DNA methylation patterns, abnormal histone modifications, and the dysregulation of non-coding RNAs, such as microRNAs (miRNAs)[3]. Azacitidine, a DNA methylation inhibitor, has shown promise by reactivating silenced tumor suppressor genes. However, its clinical application is limited due to significant side effects and the development of resistance in the advanced stages of the disease. Consequently, researchers are increasingly exploring combination therapies that can enhance the effectiveness of azacitidine while minimizing its drawbacks[10].\u003c/p\u003e\u003cp\u003eNatural compounds with well-established anticancer properties are gaining attention as potential adjuncts to conventional therapies. Oleuropein, a polyphenol present in olive leaves, exhibits diverse biological activities, encompassing anti-inflammatory, antioxidant, and anticancer effects. Preclinical studies have shown that oleuropein can promote apoptosis, block angiogenesis, and regulate critical signaling pathways, including NF-κB, PI3K/Akt, and Wnt/β-catenin[11]. Specifically, oleuropein has been shown to trigger apoptosis by enhancing the levels of caspase-3, 8, and 9, while inhibiting the PI3K-Akt pathway in the HepG2 human liver cancer cell line[12]. Research by Sophia R. Fagan et al. also found that oleuropein inhibits the growth and survival of human leukemia K562 cells by downregulating Peroxiredoxin-1 (Prdx1), a critical antioxidant in blood cells. These results highlight oleuropein as a potential adjunct to conventional cancer therapies, with the ability to enhance treatment efficacy, minimize side effects, and improve overall therapeutic outcomes[13]. ABTIN et al. reported that 600 \u0026micro;M of oleuropein was required for 50% suppression of breast cancer MCF-7 cells[14], while 170 \u0026micro;M significantly decreased cell viability in leukemia HL-60 cells. Furthermore, the IC50 of azacitidine for THP-1 and HL-60 cells has been determined to be 0.5 \u0026micro;M and 1 \u0026micro;M, respectively[9].\u003c/p\u003e\u003cp\u003eIn our study, both azacitidine and oleuropein inhibited the proliferation of THP-1 and HL-60 cells and induced apoptosis. The cytotoxic properties of these agents were assessed using the MTT assay, which revealed time-dependent effects (Table\u0026nbsp;3,4,5,6). Flow cytometry analysis, using Annexin V and propidium iodide (PI) staining, demonstrated that both oleuropein and azacitidine induced apoptotic cell death, with the highest levels of apoptosis observed 72 hours after treatment with both agents (Fig.\u0026nbsp;9,12).\u003c/p\u003e\u003cp\u003eA key feature of oleuropein\u0026rsquo;s anticancer activity is its capacity to modulate miRNA expression. MiRNAs are essential regulators of various cellular processes, such as cell proliferation, differentiation, apoptosis, and metastasis. For instance, in breast cancer cells (MCF-7), oleuropein has been shown to increase apoptosis by decreasing the expression of miR-21 and miR-155[14]. In AML, dysregulated miRNAs are commonly observed, with some acting as oncogenes and others as tumor suppressors. Given this context, investigating the impact of oleuropein on miRNA expression in AML could provide valuable insights into its potential as a therapeutic agent[6].\u003c/p\u003e\u003cp\u003eThis study explores the combined effects of oleuropein and azacitidine in AML treatment, specifically focusing on whether oleuropein can potentiate the therapeutic effects of azacitidine while minimizing its adverse effects. By examining the synergistic potential of these two compounds, our goal is to advance the development of more effective and patient-centered approaches for AML treatment. Further research is required to fully elucidate the molecular mechanisms underlying this combination and to optimize its clinical application.\u003c/p\u003e\u003cp\u003eIn this study, we examined the effects of oleuropein, both alone and in combination with azacitidine, on apoptosis and the expression of three miRNAs (miR-149-3p, miR-375, and miR-574-5p) in AML cell lines (THP-1 and HL-60). Our findings reveal critical insights into the regulatory roles of these miRNAs in AML pathogenesis and their potential therapeutic implications.\u003c/p\u003e\u003cp\u003eOur results indicate that miR-149-3p expression was significantly increased following treatment with azacitidine and oleuropein, either alone or in combination. This aligns with previous findings by MINGJUN LU et al, which reported that azacitidine upregulates miR-149-3p expression[15]. While some studies have suggested an oncogenic role for miR-149-3p in T-cell ALL[16], our findings support its tumor-suppressive function in AML, consistent with its previously reported ability to inhibit the Akt1 signaling pathway and suppress bladder cancer proliferation[17]. Notably, our study extends these findings by demonstrating that oleuropein also contributes to miR-149-3p upregulation, suggesting its potential as a therapeutic agent in AML.\u003c/p\u003e\u003cp\u003eSimilarly, our results showed a significant increase in miR-375 expression in HL-60 and THP-1 cells treated with azacitidine and oleuropein. This aligns with prior research indicating that miR-375 functions as a tumor suppressor in multiple cancers, including AML. While miR-375 has been identified as an oncogene in some cancers, such as prostate cancer and SCLC[18], our study supports its tumor-suppressive role in AML. We further demonstrated that oleuropein enhances miR-375 expression, and when combined with azacitidine, the upregulation effect was more pronounced. These findings highlight the potential of targeting the miR-375-HOXB3-CDCA3/DNMT3B regulatory axis for AML therapy[8].\u003c/p\u003e\u003cp\u003eConversely, miR-574-5p was significantly downregulated following treatment with azacitidine and oleuropein, supporting its role as a potential oncogenic miRNA in AML. Previous studies have demonstrated that miR-574-5p promotes proliferation and migration in NSCLC[19], colon cancer, and nasopharyngeal carcinoma via pathways such as β-catenin/Wnt[20]. While its role in leukemia has been less explored, our results suggest that suppressing miR-574-5p expression may be a viable therapeutic strategy. The reduction in miR-574-5p levels following oleuropein and azacitidine treatment provides new insights into its regulatory role in AML and highlights the potential of combination therapy for targeted miRNA modulation.\u003c/p\u003e\u003cp\u003eOverall, our findings underscore the importance of miRNA regulation in AML and suggest that azacitidine and oleuropein, either individually or in combination, may serve as effective modulators of miRNA expression. Future research should aim to clarify the specific molecular mechanisms driving these effects and assess their therapeutic potential in clinical environments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"5. Limitations and Future Directions","content":"\u003cp\u003eWhile our in vitro study provides compelling evidence for the synergistic effects of oleuropein and azacitidine in AML, several limitations should be addressed in future work:\u003c/p\u003e\u003cp\u003eIn Vivo Validation:\u003c/p\u003e\u003cp\u003eFuture studies should evaluate the combination therapy in animal models to assess pharmacokinetics, biodistribution, and therapeutic efficacy.\u003c/p\u003e\u003cp\u003eMechanistic Insights:\u003c/p\u003e\u003cp\u003eDetailed investigation of the molecular pathways affected by the combination treatment\u0026mdash;such as its impact on DNMT activity, histone modifications, and broader gene expression patterns using RNA-Seq\u0026mdash;is warranted.\u003c/p\u003e\u003cp\u003eClinical Translation:\u003c/p\u003e\u003cp\u003eDose optimization studies and clinical trials are needed to determine the safety, tolerability, and long-term efficacy of this combination, particularly in elderly patients and those with comorbidities.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eOur study demonstrates that the combination of oleuropein and azacitidine significantly enhances therapeutic outcomes in AML by inducing apoptosis and modulating key miRNAs (upregulating miR-149-3p and miR-375, and downregulating miR-574-5p). These findings suggest that combination therapy not only potentiates the effects of azacitidine but may also provide a less toxic alternative to high-dose chemotherapy. This research represents one of the first systematic investigations into the miRNA-modulatory effects of oleuropein in AML, paving the way for further in vivo studies and clinical trials to optimize this promising therapeutic strategy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: This study was approved by the Ethics Committee of Tabriz University of Medical Sciences, Tabriz, Iran\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor's Contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Karimi Kelaye conceived and designed the study, performed the majority of the experimental work, analyzed the data, and drafted the initial manuscript, B. Kazemi participated in data collection\u0026nbsp;and performed statistical analyses, Z.\u0026nbsp;Foruzandeh and F.Najafi were responsible for additional data acquisition, S\u0026nbsp;\u0026nbsp;Solali supervised the study, provided essential guidance, coordinated research activities, and critically revised the final manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was financially supported by the Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e: We gratefully acknowledge the staff of the Division of Hematology and Blood Banking, Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, for their valuable support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFennell KA, Bell CC, Dawson MA. Epigenetic therapies in acute myeloid leukemia: where to from here? [Internet]. 2019. Available from: https://ashpublications.org/blood/article-pdf/134/22/1891/1543146/bloodbld2019003262.pdf\u003c/li\u003e\n\u003cli\u003eIssah MA, Wu D, Zhang F, Zheng W, Liu Y, Fu H, et al. Epigenetic modifications in acute myeloid leukemia: The emerging role of circular RNAs (Review). Vol. 59, International Journal of Oncology. Spandidos Publications; 2021. \u003c/li\u003e\n\u003cli\u003eKarimi Kelaye S, Najafi F, Kazemi B, Foruzandeh Z, Seif F, Solali S, et al. The contributing factors of resistance or sensitivity to epigenetic drugs in the treatment of AML. Vol. 24, Clinical and Translational Oncology. Springer Science and Business Media Deutschland GmbH; 2022. p. 1250\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eRishmawi S, Haddad F, Dokmak G, Karaman R. A Comprehensive Review on the Anti-Cancer Effects of Oleuropein. Vol. 12, Life. MDPI; 2022. \u003c/li\u003e\n\u003cli\u003eMahi H, Yousefi Z, Toufan F, Yarmohammadi M, Jafarisani M, Eskandari N, et al. Oleuropein as An Effective Suppressor of Inflammation and MicroRNA-146a Expression in Patients with Rheumatoid Arthritis. Cell J. 2023;25(7):505\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eChandraprabha Vineetha R, Anitha Geetha Raj J, Devipriya P, Sreelatha Mahitha M, Hariharan S. MicroRNA-based therapies: Revolutionizing the treatment of acute myeloid leukemia. Vol. 46, International Journal of Laboratory Hematology. John Wiley and Sons Inc; 2024. p. 33\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003eTian Y, Jiang Y, Dong X, Chang Y, Chi J, Chen X. miR-149-3p suppressed epithelial\u0026ndash;mesenchymal transition and tumor development in acute myeloid leukemia. Hematology (United Kingdom). 2021;26(1):840\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eBi L, Zhou B, Li H, He L, Wang C, Wang Z, et al. A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia. BMC Cancer. 2018 Feb 13;18(1). \u003c/li\u003e\n\u003cli\u003eXiao X, Xu Q, Sun Y, Lu Z, Li R, Wang X, et al. 5-aza-2\u0026prime;-deoxycytidine promotes migration of acute monocytic leukemia cells via activation of CCL2-CCR2-ERK signaling pathway. Mol Med Rep. 2017 Aug 1;16(2):1417\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eWang X, Xiao Z, Qin T, Xu Z, Jia Y, Qu S, et al. Combination therapy with venetoclax and azacitidine for the treatment of myelodysplastic syndromes with DDX41 mutations. Hematology (United Kingdom). 2024;29(1). \u003c/li\u003e\n\u003cli\u003eShamshoum H, Vlavcheski F, Tsiani E. Anticancer effects of oleuropein. Vol. 43, BioFactors. Blackwell Publishing Inc.; 2017. p. 517\u0026ndash;28. \u003c/li\u003e\n\u003cli\u003eYan CM, Chai EQ, Cai HY, Miao GY, Ma W. Oleuropein induces apoptosis via activation of caspases and suppression of phosphatidylinositol 3-kinase/protein kinase B pathway in HepG2 human hepatoma cell line. Mol Med Rep. 2015 Jun 1;11(6):4617\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eOleuropein Reduces Prdx1 Expression, Cell Proliferation and Viability in K562 Human Leukemia Cells. ARC Journal of Cancer Science. 2019;5(1). \u003c/li\u003e\n\u003cli\u003eAbtin M, Alivand MR, Khaniani MS, Bastami M, Zaeifizadeh M, Derakhshan SM. Simultaneous downregulation of miR-21 and miR-155 through oleuropein for breast cancer prevention and therapy. J Cell Biochem. 2018 Sep 1;119(9):7151\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eLu M, Xu L, Wang M, Guo T, Luo F, Su N, et al. MiR-149 promotes the myocardial differentiation of mouse bone marrow stem cells by targeting Dab2. Mol Med Rep. 2018 Jun 1;17(6):8502\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eZhang M, Gao D, Shi Y, Wang Y, Joshi R, Yu Q, et al. miR-149-3p reverses CD8+ T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells. Open Biol. 2019 Oct 31;9(10):190061. \u003c/li\u003e\n\u003cli\u003eYang D, Du G, Xu A, Xi X, Li D. Expression of miR-149-3p inhibits proliferation, migration, and invasion of bladder cancer by targeting S100A4 [Internet]. Vol. 7, Am J Cancer Res. 2017. Available from: www.ajcr.us/ISSN:2156-6976/ajcr0064090\u003c/li\u003e\n\u003cli\u003eYan JW, Lin JS, He XX. The emerging role of miR-375 in cancer. Vol. 135, International Journal of Cancer. Wiley-Liss Inc.; 2014. p. 1011\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eZhou R, Zhou X, Yin Z, Guo J, Hu T, Jiang S, et al. MicroRNA-574-5p promotes metastasis of non-small cell lung cancer by targeting PTPRU. Sci Rep. 2016 Oct 20;6. \u003c/li\u003e\n\u003cli\u003eLin Z, Chen M, Wan Y, Lei L, Ruan H. miR-574-5p Targets FOXN3 to Regulate the Invasion of Nasopharyngeal Carcinoma Cells via Wnt/\u0026beta;-Catenin Pathway. Technol Cancer Res Treat. 2020;19. \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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acute myeloid leukemia, azacitidine, oleuropein, microRNA, mir-149-3p, mir-375, mir-574-5p","lastPublishedDoi":"10.21203/rs.3.rs-7091096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7091096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAcute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by the rapid expansion of immature myeloid cells and poor clinical outcomes. Despite conventional treatments, including chemotherapy and hematopoietic stem cell transplantation, relapse and resistance remain significant challenges. Epigenetic alterations, particularly dysregulated DNA methylation and microRNA (miRNA) expression, play a crucial role in AML pathogenesis.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aimed to evaluate the synergistic effects of azacitidine, a DNA methyltransferase inhibitor, and oleuropein, a natural polyphenol with anticancer properties, on AML cell lines (THP-1 and HL-60).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAML cells were treated with azacitidine, oleuropein, and their combination. Cell proliferation was assessed using MTT assays, apoptosis was analyzed via flow cytometry (Annexin V-FITC/PI staining), and miRNA expression levels were quantified using real-time PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBoth azacitidine and oleuropein reduced cell viability and induced apoptosis in a dose- and time-dependent manner. Notably, the combination treatment significantly enhanced apoptosis, with a 2.5-fold increase in Annexin V-positive HL-60 cells at 72 hours. Furthermore, the treatment modulated miRNA expression, upregulating miR-149-3p and miR-375 while downregulating miR-574-5p.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe synergistic effects of oleuropein and azacitidine suggest a potential therapeutic strategy for AML by targeting epigenetic mechanisms and miRNA pathways. Further in vivo studies and clinical trials are necessary to validate these findings and optimize treatment protocols.\u003c/p\u003e","manuscriptTitle":"Combined Effects of 5-Azacytidine and Oleuropein on miR-149-3p, miR-375, miR-574-5p Expression and Apoptosis in HL-60 and THP-1 Cell Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 14:09:18","doi":"10.21203/rs.3.rs-7091096/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-04T14:32:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T23:22:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T11:12:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134198094615792519776317578354326703298","date":"2025-07-15T04:15:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18314541758286328082862824141656332953","date":"2025-07-14T12:43:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T12:19:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-12T11:11:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-12T11:11:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-07-10T09:02:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"38f3d88a-8d15-4db7-87f3-7d4b72fb4699","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:09:20+00:00","versionOfRecord":{"articleIdentity":"rs-7091096","link":"https://doi.org/10.1007/s11033-025-11068-z","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-10-17 15:57:54","publishedOnDateReadable":"October 17th, 2025"},"versionCreatedAt":"2025-07-17 14:09:18","video":"","vorDoi":"10.1007/s11033-025-11068-z","vorDoiUrl":"https://doi.org/10.1007/s11033-025-11068-z","workflowStages":[]},"version":"v1","identity":"rs-7091096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7091096","identity":"rs-7091096","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}