Diabetic Foot: A MicroRNA-Centric Approach

Diabetic Foot: A MicroRNA-Centric Approach | 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 Diabetic Foot: A MicroRNA-Centric Approach Luís Matos de Oliveira, Gabriela Correia Matos de Oliveira, Tulio Matos David, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4278543/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introdução: Diabetic neuropathy-associated vasculopathy is a significant risk factor for the development of diabetic foot ulcers (DFUs). In the context of DFUs, miRNAs can influence the cascade of molecular events that culminate in healing. Objective : To design in silico the molecular structures of microRNAs (miRNAs) overexpressed in diabetic foot ulcer healing. Methods : We conducted a search for the nucleotide sequences of eight miRNAs overexpressed in DFUs, and the following miRNAs were selected: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. These miRNAs were selected for evaluation in this study based on pre-clinical evidence, differential expression in DFUs, and therapeutic potential. Subsequently, the molecular structures of the eight miRNAs were designed in silico . The nucleotide sequences were retrieved from GenBank, the genetic sequence database of the National Center for Biotechnology Information. The obtained sequences were aligned using multiple alignment algorithms from the RNA Fold web server. RNAComposer, an automated miRNA structure modeling server, was employed for the in silico modeling of the structures. Results : We performed a search for the nucleotide sequences and designed the molecular structures of the following miRNAs overexpressed in diabetic foot ulcer healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. We generated a tutorial on the molecular models of these eight miRNAs overexpressed in the diabetic foot, based on in silico projections of their molecular structures. Conclusion : This study demonstrates the in silico design of secondary structures for a selection of eight miRNAs overexpressed in diabetic foot ulcer healing, utilizing techniques from computational biology. Endocrinology & Metabolism Bioinformatics Diabetic foot microRNA Molecular structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 INTRODUCTION Diabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus, arising from a complex interplay of peripheral vascular disease and diabetic neuropathy, leading to deep tissue ulcerations in the lower extremities. Epidemiological studies report that approximately 15% of diabetic patients develop DFUs, with 85% of all amputations due to diabetic foot issues being preceded by ulceration, potentially progressing to infection or gangrene. 1 , 2 The pathophysiology of DFUs is rooted in neuropathy, trauma, and, in some cases, co-existing occlusive arterial disease of the lower limbs, all of which significantly elevate the risk of ulceration with concomitant soft tissue infection. 3 miRNA are short, non-coding RNA molecules (19–35 nucleotides) that function as crucial regulatory elements in post-transcriptional gene expression within multicellular organisms. 4 Recent years have witnessed a dramatic increase in our understanding of miRNA structure and function. 5 Bioinformatics software advancements have provided researchers with powerful tools for constructing molecular models and analyzing nucleotide sequences, enabling a deeper exploration of miRNA-mediated molecular mechanisms. 6 This study aimed to utilize in silico techniques to design the three-dimensional (3D) molecular structures of eight miRNA known to be overexpressed in DFUs. Furthermore, we sought to develop a tutorial on the modeling process employed. METHODS Nucleotide Sequence Retrieval and Alignment To acquire the nucleotide sequences of the miRNAs under investigation, we utilized the National Center for Biotechnology Information's (NCBI) GenBank genetic sequence database ( https://www.ncbi.nlm.nih.gov/ ). Following sequence retrieval, we employed the RNA Fold web server ( http://www.unafold.org/ ), for sequence alignment. Subsequently, 3D molecular structures of the miRNAs were modeled using RNAComposer ( https://rnacomposer.cs.put.poznan.pl/ ). Finally, by leveraging in silico approaches, we constructed a comprehensive tutorial showcasing the predicted molecular arrangements of the eight miRNAs. The target miRNA included: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. They were selected for evaluation in the diabetic foot study for several reasons: preclinical studies have demonstrated that these miRNAs are involved in regulating various molecular processes important for wound healing, comparative gene expression studies have shown that these miRNAs are differentially expressed in DFUs compared to non-wounded tissue, and modulation of the expression of these miRNAs may be a promising strategy for the treatment of DFUs. Tutorial Development This study involved the creation of a tutorial that guides users through the process of constructing molecular models for the eight miRNA overexpressed in the diabetic foot. The tutorial leverages in silico projections of the modeled miRNA structures. Methods Descriptions Nucleotide Search and Sequence Analysis: GenBank is a publicly accessible database for nucleotide sequence analysis. It offers various search algorithms and a comprehensive collection of databases encompassing diverse nucleotide sequences. Notably, GenBank incorporates the Nucleotide, Genome Survey Sequence (GSS), and Expressed Sequence Tag (EST) databases, which collectively house a vast array of nucleic acid sequences. Information within EST and GSS originates from sequencing data shared through GenBank. Molecular Model Building The function and structural integrity of amino acids and proteins are fundamentally determined by their specific sequence of nucleotides. Consequently, high-resolution structure determination methods play a crucial role in elucidating their 3D organization. Among computational approaches, homology modeling stands out as the most accurate technique for generating reliable structural models. This method's versatility makes it a prominent tool across various biological research areas. Modeling RNA Structure Leveraging the principles of machine translation, RNAComposer automates the prediction of RNA 3D structures. This interactive system utilizes the RNA FRABASE database as a reference for translating secondary structures into corresponding tertiary arrangements. While RNAComposer permits analysis of RNA molecules up to 500 nucleotides in length, it currently generates a single predicted 3D model per analysis. The software is freely available for public download from the developers' website: https://rnacomposer.cs.put.poznan.pl/references . Ethical Considerations In accordance with Resolution CNS 510/2016 of the National Health Council (CNS), this study did not require evaluation by the Research Ethics Committee. This is because the research focused on the theoretical investigation of the understanding of pathologies in clinical practice through the use of bioinformatics tools. RESULTS This section presents the results of in silico modeling for the molecular structures of eight miRNAs known to be overexpressed in diabetic foot ulcer healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. Additionally, a tutorial was developed to guide users in constructing molecular models for these miRNAs based on in silico projections of their structures. MiRNA-146a Nucleotide Sequence The nucleotide sequence for miRNA-146a (NCBI accession: NR_029701.1) was retrieved from GenBank in FASTA format. This sequence encodes a 99-nucleotide linear molecule. The analysis for Homo sapiens microRNA 146a (MIR146A) is presented in Fig. 1 . Molecular Model The nucleotide sequence for miRNA-146a (Homo sapiens microRNA 146A, MIR146A) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-146a. Based on this alignment, a structural model for miRNA-146a was generated using comparative nucleotide modeling on the RNAComposer (Fig. 2 ). MiRNA-155 Nucleotide Sequence The nucleotide sequence for miRNA-155 (NCBI Reference Sequence: NR_030784.1) was retrieved from the GenBank database in FASTA format. This sequence encodes a 65-nucleotide linear non-coding RNA (ncRNA). All FASTA-formatted sequences were obtained with annotations from NCBI - Graphics. The analysis for Homo sapiens microRNA 155 (MIR155) is shown in Fig. 3 . Molecular Model The structural template for miRNA-155 was constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 155) and the template structure. Assessment tools were employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 155 was generated using comparative nucleotide modeling on the RNAComposer (Fig. 4 ). MiRNA-132 Nucleotide Sequence The nucleotide sequence for miRNA-132 (NCBI accession: NR_029674.1) was retrieved from GenBank in FASTA format. This sequence encodes a 101-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 132 (MIR132) (Fig. 5 ). Molecular Model The nucleotide sequence for miRNA-132 (Homo sapiens microRNA 132, MIR132) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-132. Based on this alignment, a structural model for miRNA-132 was generated using comparative nucleotide modeling on the RNAComposer (Fig. 6 ). MiRNA-191 Nucleotide Sequence An inconsistency exists in the provided information for miRNA-191. The NCBI accession code for miRNA-132 (NR_029690.1) is used here, while the figure caption likely refers to a different miRNA. Please verify and correct the nucleotide sequence and NCBI accession code for miRNA-191. The analysis for the corrected miRNA-191 will be presented in Fig. 7 . Molecular Model Following the acquisition of the correct nucleotide sequence for miRNA-191, the structural template will be constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 191) and the template structure. Assessment tools will be employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 191 will be generated using comparative nucleotide modeling on the RNAComposer (Fig. 8 ). MiRNA-21 Nucleotide Sequence The nucleotide sequence for miRNA-21 (NCBI accession: NR_029493.1) was retrieved from GenBank in FASTA format. This sequence encodes a 72-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 21 (MIR21) is demonstrated in Fig. 9 . Molecular Model The nucleotide sequence for miRNA-21 (Homo sapiens microRNA 21, MIR21) was acquired employing FASTA format; modeling was performed employing the RNAComposer, optimized and adjusted for alignment between structural templates and miRNA-21 nucleotide. Based on sequence alignment between the template structure and miRNA-21 nucleotide, a structural model was built for the nucleotide in question. So, employing the RNAComposer of comparative nucleotide modeling, we generated a homology model of microRNA 21 (MIR21), demonstrated in Fig. 10 . MiRNA-203a Nucleotide Sequence The nucleotide sequence for miRNA-203a (NCBI accession: NR_029620.1) was retrieved from GenBank in FASTA format. This sequence encodes a 110-bp linear ncRNA. The analysis for Homo sapiens microRNA 203a (MIR203A) (Fig. 11 ). Molecular Model The nucleotide sequence for miRNA-203a (Homo sapiens microRNA 203a, MIR203A) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-203a. Based on this alignment, a structural model for miRNA-203a was generated using comparative nucleotide modeling on the RNAComposer (Fig. 12 ). MiRNA-203b Nucleotide Sequence The nucleotide sequence for miRNA-203b (NCBI accession: NR_039859.1) was retrieved from GenBank in FASTA format. This sequence encodes an 86-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 203b (MIR203B) is demonstrated in Fig. 13 . Molecular Model The nucleotide sequence for miRNA-203b (Homo sapiens microRNA 203b, MIR203B) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-203b. Based on this alignment, a structural model for miRNA-203b was generated using comparative nucleotide modeling on the RNAComposer (Fig. 14 ). MiRNA-210 Nucleotide Sequence The nucleotide sequence for miRNA-210 (NCBI accession: NR_029623.1) was retrieved from GenBank in FASTA format. This sequence encodes a 110-bp linear ncRNA. The analysis for Homo sapiens microRNA 210 (MIR210) is shown in Fig. 15 . Molecular Model The structural template for miRNA-210 was constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 210) and the template structure. Assessment tools were employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 210 was generated using comparative nucleotide modeling on the RNAComposer (Fig. 16 ). DISCUSSION miRNA are small, non-coding RNA that play critical roles in various physiological and pathophysiological processes. Over the past decade, our understanding of miRNA structure and function has advanced significantly. This study presents a tutorial on in silico modeling of miRNA structures and demonstrates the predicted 3D structures of eight miRNAs known to be overexpressed in DFUs. DFUs are a severe complication of diabetes mellitus, contributing significantly to morbidity and mortality. Understanding the underlying genetic and molecular mechanisms is crucial for improving treatment efficacy. Various miRNA and their target genes are involved in both tissue fibrosis and angiogenesis in DFUs. 6 , 7 Located on chromosome 5q33.3, miRNA-146a is implicated in the pathogenesis of many diseases by inhibiting target gene expression. The miRNA-146a family (including miRNA-146a and miRNA-146b) negatively regulates inflammatory gene expression in monocytes, endothelial cells, and epithelial cells. Additionally, miRNA-146a induces the innate immune response upon encountering antigens. 8 While miRNA-146a expression is significantly reduced in DFUs themselves, studies show increased expression in surrounding tissues, suggesting context-dependent regulation. 9 Interestingly, treatment with a miR-146a inhibitor promotes faster wound healing in DFUs, potentially through increased vascular endothelial growth factor and fibronectin production. 10 The post-genomic era has witnessed significant technological advancements, leading to the expansion of microarrays and databases. However, a major challenge for researchers lies in extracting valuable information from this vast resource. Bioinformatics offers a multitude of tools for predicting miRNA structures, which can ultimately lead to a deeper understanding of miRNA transcriptional patterning. In this study, we investigated the sequence of miRNA-146a using the NCBI database, specifically the GenBank resource identified by the corresponding NCBI identifier. We then employed bioinformatics tools to analyze the encoded nucleotides within miRNA-146a and subsequently modeled its structure. Bioinformatics tools for miRNA prediction have become increasingly accessible due to the relative difficulty of performing experimental miRNA structure determination. In silico evaluation of miRNAs primarily relies on the analysis of their primary and secondary structures. Our literature review identified no prior studies presenting a 3D structure model for miRNA-146a. To address this gap, we employed the GenBank nucleotide database to retrieve the miRNA-146a sequence and subsequently utilized the RNAComposer online program to generate its predicted 3D model. MiRNA-155 is a small, single-stranded, non-coding RNA located at chromosome 21q21.3 in humans. Previously known as B-cell Integration Cluster, it is encoded by the MIR155 host gene (MIR155HG). MiRNA-155 plays a diverse role in various physiological and pathological processes. Studies suggest its potential involvement in the pathogenesis of diabetic complications. 11 , 12 Treating chronic DFUs presents a significant challenge due to the conflicting requirements of managing inflammation and promoting re-epithelialization. Both processes can contribute to increased bacterial colonization, ultimately delaying wound healing. Research has shown an association between miR-155 and increased severity of DFUs. 4 Diabetic patients often exhibit reduced levels of miRNA-155 expression in peripheral blood mononuclear cells. Conversely, inhibition of miRNA-155 leads to decreased inflammation, possibly due to increased M2 macrophage polarization and type I collagen deposition. 13 Additionally, in immune cells, miRNA-155 plays a pro-inflammatory role by suppressing cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). 14 A literature review identified only one prior study presenting a 3D structural model of miRNA-155. 15 In this study, we employed a computational approach to analyze the miRNA-155 sequence and predict its 3D structure. We retrieved the sequence using the FASTA format from a nucleotide database. Subsequently, the RNAComposer program was utilized for 3D modeling. The program was optimized to achieve alignment between structural templates and the miRNA-155 sequence. This approach, based on structural homology, facilitated the generation of the predicted 3D model of miRNA-155. The development of accurate molecular models holds promise for advancing the development of novel therapeutic drugs that target specific organs. miRNA-132 is a microRNA transcribed from an intergenic region on human chromosome 17 at location 17p13.3. 16 It targets various factors involved in angiogenesis and inflammation, both of which are crucial processes in DFUs. miRNA-132 can promote endothelial cell proliferation and contribute to neovascularization through angiogenic factors. 17 Conversely, miRNA-132 can also modulate inflammation, potentially contributing to the underlying inflammation associated with diabetic insulin resistance. 18 Studies have shown the efficacy of miRNA-132 in promoting wound healing in diabetic mouse models, and a case study suggests its potential for topical application in human diabetic wounds. 19 Our literature review did not identify any published studies presenting a 3D structural model of miRNA-132. Therefore, we utilized a homology modeling approach based on the miRNA-132 sequence retrieved from the NCBI Nucleotide database. The RNAComposer web server was employed to generate a predicted 3D model of miRNA-132 using a high-homology structure as a template. miRNA-191, a member of the miR-191 family, is located on human chromosome 3 at position 3p21.31. 20 Its expression modulates cell proliferation, apoptosis, and the cell cycle in various diseases. Additionally, miRNA-191 is a stress-sensitive miRNA, and low levels have been linked to diabetes mellitus. 21 A recent study demonstrated altered plasma miRNA-191 profiles in diabetic patients with impaired wound healing compared to diabetic patients without chronic foot ulcers, suggesting a potential role in wound healing. 22 Our literature review identified no published secondary structure models for miRNA-191. Therefore, we employed a computational homology modeling approach to predict the 3D structure of Homo sapiens miRNA-191. The FASTA sequence of miRNA-191 was used in conjunction with a high-homology structure as a template on the RNAComposer web server to generate the predicted 3D model. Located within a coding gene for vacuole membrane protein on chromosome 17q23.2, human miRNA-21 plays a critical role in various diseases, biological development, and regulation of immunological processes. 23 Studies have shown that miR-21 expression is downregulated during diabetic wound healing. MiRNA-21 is crucial for fibroblast migration, collagen deposition, and exhibits pro-proliferative, anti-apoptotic, and anti-inflammatory properties. 24 Its expression varies throughout the diabetic wound healing process, with high levels in the inflammatory phase, decreasing during the proliferative phase, and significantly increasing during the remodeling phase. 25 Our literature review identified only one published study presenting a 3D structural model of miRNA-21. 26 Therefore, we employed the FASTA sequence of miRNA-21 retrieved from the GenBank nucleotide database to generate a predicted 3D model using the RNAComposer web server. miRNA-203a and 203b, located on human chromosome 14q32.33, play a role in modulating the inflammatory response and cell proliferation, particularly important for skin development. 27 Studies have shown increased expression levels of miRNA-203 in DFUs. Notably, miRNA-203 is highly abundant in the epidermis and may play a significant role in diabetic wound healing. However, research suggests a positive correlation between miRNA-203 expression and diabetic foot ulcer severity. 28 Our literature review identified no published studies presenting 3D structural models for either miRNA-203a or miRNA-203b. Therefore, we utilized the GenBank nucleotide database to retrieve the sequences of both miRNAs and subsequently employed the RNAComposer web server to generate predicted 3D models for miRNA-203a and miRNA-203b. miRNA-210, located on chromosome 11p15.5, is involved in various biological processes within the human body, regulating cellular functions such as metabolism, cell proliferation, apoptosis, and angiogenesis. 29 Studies have shown reduced expression of miR-210 in DFUs. Local reconstitution using miR-210 mimics significantly improves diabetic wound healing, potentially by decreasing the wound's oxygen consumption rate through a local reduction of reactive oxygen species (ROS) levels in the wound tissue. 30 Our literature review did not identify any published studies presenting a secondary structure model for miRNA-210. Therefore, we employed a computational approach to build a predicted 3D structure model for Homo sapiens miRNA-210. The FASTA sequence of miRNA-210 was used in conjunction with a high-homology structure as a template on the RNAComposer web server to generate the model. CONCLUSION miRNAs are small non-coding RNAs with essential functions in various biological processes. Their structure and function are determined by their nucleotide sequence. Computational structure prediction methods can be employed to identify potential binding sites within these nucleotides, which have significant value for clinical and pharmacological applications. This study used in silico approaches to analyze the secondary structures of eight selected miRNAs, known to be overexpressed in DFUs, and modulation of the expression of these miRNAs may be a promising strategy for the treatment of DFUs. DECLARATIONS Disclosure of potential conflicts of interest: None of the authors have any potential conflicts of interest to disclose. REFERENCES Zhang P, Lu J, Jing Y, Tang S, Zhu D, Bi Y. Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis. Ann Med. 2017;49(2):106-116. Brocco E, Ninkovic S, Marin M, et al. Diabetic foot management: multidisciplinary approach for advanced lesion rescue. J Cardiovasc Surg (Torino). 2018;59(5):670-684. Bandyk DF. The diabetic foot: Pathophysiology, evaluation, and treatment. Semin Vasc Surg. 2018;31(2-4):43-48. Moura J, Sørensen A, Leal EC, et al. microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation. Sci Rep. 2019;9(1):5836. Saliminejad K, Khorram Khorshid HR, Soleymani Fard S, Ghaffari SH. Na overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol. 2019;234(5):5451-5465. Liang L, Stone RC, Stojadinovic O, et al. Integrative Analysis of miRNA and mRNA Paired Expression Profiling of Primary Fibroblast Derived from Diabetic Foot Ulcers Reveals Multiple Impaired Cellular Functions. Wound Repair Regen. 2016;24(6):943-953. Meng S, Cao J, Zhang X, et al. Downregulation of microRNA-130a contributes to endothelial progenitor cell dysfunction in diabetic patients via its target Runx3. PLoS One. 2013;8(7):e68611. Wang H, Li X, Li T, et al. Oncol Lett. 2019;18(5):5033-5042. Bruhn-Olszewska B, Korzon-Burakowska A, Gabig-Cimińska M, Olszewski P, Węgrzyn A, Jakóbkiewicz-Banecka J. Molecular factors involved in the development of diabetic foot syndrome. Acta Biochim Pol. 2012;59(4):507-13. Feng B, Chen S, Zhang L, Cao Y, Chakrabarti S. miRNA-146a and miRNA200b Antagomirs Accelerate Wound Healing through the Regulation of VEGF and Fibronectin. Journal of Pharmacy and Pharmacology. 2014;2:104-113. Faraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: a typical multifunctional microRNA. Biochim Biophys Acta. 2009;1792(6):497-505. Khamaneh AM, Alipour MR, Sheikhzadeh Hesari F, Ghadiri Soufi F. A signature of microRNA-155 in the pathogenesis of diabetic complications. J Physiol Biochem. 2015;71(2):301-9. Guo Y, Lin C, Xu P, et al. AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes. Sci Rep. 2016;6:36416. Zhang Y, Sun E, Li X, et al. miR-155 contributes to Df1-induced asthma by increasing the proliferative response of Th cells via CTLA-4 downregulation. Cell. Immunol. 2017;314:1–9. Renrick AN, Thounaojam MC, de Aquino MTP, et al. Bortezomib Sustains T Cell Function by Inducing miR-155-Mediated Downregulation of SOCS1 and SHIP1. Front Immunol. 2021;12:607044. Salta E, De Strooper B. microRNA-132: a key noncoding RNA operating in the cellular phase of Alzheimer's disease. FASEB J. 2017;31(2):424-433. Gougelet A, Pissaloux D, Besse A, et al. Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response. Int J Cancer. 2011;129(3):680-90. Strum JC, Johnson JH, Ward J, et al. MicroRNA 132 regulates nutritional stressinduced chemokine production through repression of SirT1. Mol Endocrinol. 2009;23(11):1876-84. Li X, Li D, Wang A, et al. MicroRNA-132 with Therapeutic Potential in Chronic Wounds. J Invest Dermatol. 2017;137(12):2630-2638. MIR191 microRNA 191 [Homo sapiens (human)]. Available from: https://www.ncbi.nlm.nih.gov/gene/406966 (accessed March 24, 2024). Nagpal N, Kulshreshtha R. miR-191: an emerging player in disease biology. Front Genet. 2014;5:99. Dangwal S, Stratmann B, Bang C, et al. Impairment of Wound Healing in Patients With Type 2 Diabetes Mellitus Influences Circulating MicroRNA Patterns via Inflammatory Cytokines. Arterioscler Thromb Vasc Biol. 2015;35(6):1480-8. Kumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and disease. RNA Biol. 2011;8(5):706-13. Tana C, Giamberardino MA, Cipollone F. microRNA profiling in atherosclerosis, diabetes, and migraine. Ann Med. 2017;49(2):93-105. Shang YY, Fang NN, Wang F, et al. MicroRNA-21, induced by high glucose,modulates macrophage apoptosis via programmed cell death 4. Mol Med Rep. 2015;12(1):463-9. Baisden JT, Boyer JA, Zhao B, Hammond SM, Zhang Q. Visualizing a protonated RNA state that modulates microRNA-21 maturation. Nat Chem Biol. 2021;17(1):80-88. Zang J, Hui L, Yang N, Yang B, Jiang X. Downregulation of MiR-203a Disinhibits Bmi1 and Promotes Growth and Proliferation of Keratinocytes in Cholesteatoma. Int J Med Sci. 2018;15(5):447-455. Liu J, Xu Y, Shu B, et al. Quantification of the differential expression levels of microRNA-203 in different degrees of diabetic foot. Int J Clin Exp Pathol. 2015;8(10):13416-20. Bavelloni A, Ramazzotti G, Poli A, et al. MiRNA-210: A Current Overview. Anticancer Res. 2017;37(12):6511-6521. Modaghegh MHS, Saberianpour S, Amoueian S, Shahri JJ, Rahimi H. The effect of redox signaling on extracellular matrix changes in diabetic wounds leading to amputation. Biochem Biophys Rep. 2021;26:101025. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4278543","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292098012,"identity":"b2a0f395-5dd1-40fa-a4d2-bb766ec3d05e","order_by":0,"name":"Luís Matos de Oliveira","email":"","orcid":"https://orcid.org/0000-0003-4854-6910","institution":"Bahiana School of Medicine and Public Health - Salvador - Bahia - Brazil.","correspondingAuthor":false,"prefix":"","firstName":"Luís","middleName":"Matos","lastName":"de Oliveira","suffix":""},{"id":292098013,"identity":"a2b79135-0bf9-4120-b5ae-df3657351a06","order_by":1,"name":"Gabriela Correia Matos de Oliveira","email":"","orcid":"https://orcid.org/0000-0002-8042-0261","institution":"Medical Doctor, UniFTC Medical School - Salvador - Bahia - Brazil.","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Correia Matos","lastName":"de Oliveira","suffix":""},{"id":292098014,"identity":"f0ce81a7-e3e6-44b6-a5fc-41240acd57b8","order_by":2,"name":"Tulio Matos David","email":"","orcid":"https://orcid.org/0009-0000-0257-5017","institution":"Federal University of Bahia School of Medicine – Salvador – Bahia – Brazil.","correspondingAuthor":false,"prefix":"","firstName":"Tulio","middleName":"Matos","lastName":"David","suffix":""},{"id":292098015,"identity":"9e238c06-60b0-4e1e-bad6-68a46d3ba3c5","order_by":3,"name":"Roseanne Montargil Rocha","email":"","orcid":"https://orcid.org/0000-0001-5766-413X","institution":"Health Department State University of Santa Cruz - Ilhéus – Bahia – Brazil.","correspondingAuthor":false,"prefix":"","firstName":"Roseanne","middleName":"Montargil","lastName":"Rocha","suffix":""},{"id":292098016,"identity":"4e5ea282-7fbe-457a-ac7d-7caa0fe55006","order_by":4,"name":"Luís Jesuíno de Oliveira Andrade","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACPgbGBgYJIIONgYHxAVhIgoAWNiQtzAZEakFiSxCnRSK5+YNFRV1in3TzsWqeijp5BuneBwS0JLZJSJw5nNgmcyztNs+Zw4YNMscNCGphkGw7ANSYY3abt+1AAoNEGiGHJTZ/kPxXB9ZSzPuvjigtDRKSDcxgLcy8DcxEaOF5CPTLscPGbRJpyZJzjh02BHoKvxZ+9vTHnyVq6mTnz0g++OFNTZ08v3Qbfi0gwIwSE2y4lCEDxg/EqBoFo2AUjIKRCwDSczuoafAOIAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7714-0330","institution":"Health Department State University of Santa Cruz, Ilhéus – Bahia – Brazil.","correspondingAuthor":true,"prefix":"","firstName":"Luís","middleName":"Jesuíno de Oliveira","lastName":"Andrade","suffix":""}],"badges":[],"createdAt":"2024-04-17 01:02:09","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4278543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4278543/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54889440,"identity":"8c0cb892-1d92-4d4a-97be-247ebf8ad88e","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15332,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens microRNA-146a – \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource:\u003c/strong\u003e \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=jZShVBlPae\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=jZShVBlPae\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/abfbde48f66ee30585db170c.png"},{"id":54889426,"identity":"887f45e0-a967-483f-85a0-474513318e62","added_by":"auto","created_at":"2024-04-18 07:18:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81057,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-146A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource:\u003c/strong\u003e \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/\"\u003ehttps://rnacomposer.cs.put.poznan.pl/\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/3e9ec48ff26f71911d5028ea.png"},{"id":54889423,"identity":"d2d27f9b-0587-4065-b5e2-e7566e10c5d7","added_by":"auto","created_at":"2024-04-18 07:18:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9396,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens microRNA-155 – \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mLPPdAVSUK\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mLPPdAVSUK\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/f2d1b8a0c9e9bea807a39465.png"},{"id":54889430,"identity":"e130f5f5-39f5-49c2-9f6c-04231ea1b13a","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":82326,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-155\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/\"\u003ehttps://rnacomposer.cs.put.poznan.pl/\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/bff76cbdf09ee39c6959a18b.png"},{"id":54889443,"identity":"7e67ea96-fe49-45b5-8016-1802670468ee","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13431,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens microRNA-132 - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mIEsZButAk\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mIEsZButAk\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/464ceb1836bcba54dadad856.png"},{"id":54889412,"identity":"201a82b8-275e-41ae-bef1-954facf68fb4","added_by":"auto","created_at":"2024-04-18 07:18:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86789,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-132\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/673c8f3b2b3145caa0753883.png"},{"id":54889434,"identity":"ba3084b0-eab7-47f7-b3a7-91fe50bd3017","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12714,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens miRNA-191 - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=TWLqxr3InL\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=TWLqxr3InL\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/df69cdb893b3f7bcc719155a.png"},{"id":54889441,"identity":"82969f00-9fb9-4474-bb25-6556e27517bc","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":85369,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-191\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/267ba0cd7fa181e62a2f9766.png"},{"id":54889445,"identity":"334367e0-606f-4f34-a2cf-0375b05743ab","added_by":"auto","created_at":"2024-04-18 07:18:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":10326,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens miRNA-21 - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=oGDR8njoT2\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=oGDR8njoT2\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/b6ad48ee325d3b03b7487c25.png"},{"id":54889438,"identity":"853f20ba-5990-4759-9c41-a4ce2a3537f3","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":42740,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-21\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/f06da5951e6b04188c49db78.png"},{"id":54889432,"identity":"078843f5-d8b2-46ea-940d-208deabe86d5","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":15679,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens miRNA-203a - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mIEsZButAk\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=mIEsZButAk\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/8526c7ccf3f707cf3ced36e9.png"},{"id":54889446,"identity":"e4a3ddf9-142d-4d73-ae2c-c4647a6e2fa5","added_by":"auto","created_at":"2024-04-18 07:18:39","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":86153,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-203a\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/c935b8715eea0679664522b5.png"},{"id":54889415,"identity":"8c681229-e938-4e7b-89a9-55d2818c1f24","added_by":"auto","created_at":"2024-04-18 07:18:37","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":13487,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens miRNA-203b - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=eG73WRwb66\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=eG73WRwb66\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/0fd84f8062abf165501595a8.png"},{"id":54889436,"identity":"762ff5d9-6f01-4ef0-9a00-35e281b2612f","added_by":"auto","created_at":"2024-04-18 07:18:38","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":59761,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-203b\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/cde3e9757b9b666308c2f3c6.png"},{"id":54890081,"identity":"b452ab65-376d-475a-8308-dd5bfb41f256","added_by":"auto","created_at":"2024-04-18 07:26:38","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":15825,"visible":true,"origin":"","legend":"\u003cp\u003eHomo sapiens miRNA-210 - \u003cem\u003emodel-template aligment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=8dlyu9CPDK\"\u003ehttp://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3\u0026amp;ID=8dlyu9CPDK\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/e37bbad4506ab49555afcee9.png"},{"id":54889429,"identity":"ee9ec6c2-2109-4a5c-adff-7f38da136d21","added_by":"auto","created_at":"2024-04-18 07:18:37","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":80967,"visible":true,"origin":"","legend":"\u003cp\u003eHomology model of Homo sapiens microRNA-210\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e: \u003ca href=\"https://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\"\u003ehttps://rnacomposer.cs.put.poznan.pl/;jsessionid=EB25C57322A58CB0C21EA783CDF6FD81\u003c/a\u003e\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/34354147eba1e4f5c7c12168.png"},{"id":54890083,"identity":"34a06407-dab0-4685-9e90-358b581d2a01","added_by":"auto","created_at":"2024-04-18 07:26:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1296657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4278543/v1/3e6aa74d-e6fe-4c38-a978-f5e58d043a08.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDiabetic Foot: A MicroRNA-Centric Approach\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDiabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus, arising from a complex interplay of peripheral vascular disease and diabetic neuropathy, leading to deep tissue ulcerations in the lower extremities. Epidemiological studies report that approximately 15% of diabetic patients develop DFUs, with 85% of all amputations due to diabetic foot issues being preceded by ulceration, potentially progressing to infection or gangrene.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The pathophysiology of DFUs is rooted in neuropathy, trauma, and, in some cases, co-existing occlusive arterial disease of the lower limbs, all of which significantly elevate the risk of ulceration with concomitant soft tissue infection.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003emiRNA are short, non-coding RNA molecules (19\u0026ndash;35 nucleotides) that function as crucial regulatory elements in post-transcriptional gene expression within multicellular organisms.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Recent years have witnessed a dramatic increase in our understanding of miRNA structure and function.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Bioinformatics software advancements have provided researchers with powerful tools for constructing molecular models and analyzing nucleotide sequences, enabling a deeper exploration of miRNA-mediated molecular mechanisms.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study aimed to utilize \u003cem\u003ein silico\u003c/em\u003e techniques to design the three-dimensional (3D) molecular structures of eight miRNA known to be overexpressed in DFUs. Furthermore, we sought to develop a tutorial on the modeling process employed.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNucleotide Sequence Retrieval and Alignment\u003c/h2\u003e \u003cp\u003eTo acquire the nucleotide sequences of the miRNAs under investigation, we utilized the National Center for Biotechnology Information's (NCBI) GenBank genetic sequence database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Following sequence retrieval, we employed the RNA Fold web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.unafold.org/\u003c/span\u003e\u003cspan address=\"http://www.unafold.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), for sequence alignment. Subsequently, 3D molecular structures of the miRNAs were modeled using RNAComposer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rnacomposer.cs.put.poznan.pl/\u003c/span\u003e\u003cspan address=\"https://rnacomposer.cs.put.poznan.pl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, by leveraging \u003cem\u003ein silico\u003c/em\u003e approaches, we constructed a comprehensive tutorial showcasing the predicted molecular arrangements of the eight miRNAs.\u003c/p\u003e \u003cp\u003eThe target miRNA included: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. They were selected for evaluation in the diabetic foot study for several reasons: preclinical studies have demonstrated that these miRNAs are involved in regulating various molecular processes important for wound healing, comparative gene expression studies have shown that these miRNAs are differentially expressed in DFUs compared to non-wounded tissue, and modulation of the expression of these miRNAs may be a promising strategy for the treatment of DFUs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTutorial Development\u003c/h2\u003e \u003cp\u003eThis study involved the creation of a tutorial that guides users through the process of constructing molecular models for the eight miRNA overexpressed in the diabetic foot. The tutorial leverages \u003cem\u003ein silico\u003c/em\u003e projections of the modeled miRNA structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMethods Descriptions\u003c/h2\u003e \u003cp\u003eNucleotide Search and Sequence Analysis: GenBank is a publicly accessible database for nucleotide sequence analysis. It offers various search algorithms and a comprehensive collection of databases encompassing diverse nucleotide sequences. Notably, GenBank incorporates the Nucleotide, Genome Survey Sequence (GSS), and Expressed Sequence Tag (EST) databases, which collectively house a vast array of nucleic acid sequences. Information within EST and GSS originates from sequencing data shared through GenBank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Model Building\u003c/h2\u003e \u003cp\u003eThe function and structural integrity of amino acids and proteins are fundamentally determined by their specific sequence of nucleotides. Consequently, high-resolution structure determination methods play a crucial role in elucidating their 3D organization. Among computational approaches, homology modeling stands out as the most accurate technique for generating reliable structural models. This method's versatility makes it a prominent tool across various biological research areas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eModeling RNA Structure\u003c/h2\u003e \u003cp\u003eLeveraging the principles of machine translation, RNAComposer automates the prediction of RNA 3D structures. This interactive system utilizes the RNA FRABASE database as a reference for translating secondary structures into corresponding tertiary arrangements. While RNAComposer permits analysis of RNA molecules up to 500 nucleotides in length, it currently generates a single predicted 3D model per analysis. The software is freely available for public download from the developers' website: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rnacomposer.cs.put.poznan.pl/references\u003c/span\u003e\u003cspan address=\"https://rnacomposer.cs.put.poznan.pl/references\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthical Considerations\u003c/h2\u003e \u003cp\u003eIn accordance with Resolution CNS 510/2016 of the National Health Council (CNS), this study did not require evaluation by the Research Ethics Committee. This is because the research focused on the theoretical investigation of the understanding of pathologies in clinical practice through the use of bioinformatics tools.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThis section presents the results of \u003cem\u003ein silico\u003c/em\u003e modeling for the molecular structures of eight miRNAs known to be overexpressed in diabetic foot ulcer healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. Additionally, a tutorial was developed to guide users in constructing molecular models for these miRNAs based on \u003cem\u003ein silico\u003c/em\u003e projections of their structures.\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-146a\u003c/h2\u003e\n\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-146a (NCBI accession: NR_029701.1) was retrieved from GenBank in FASTA format. This sequence encodes a 99-nucleotide linear molecule. The analysis for Homo sapiens microRNA 146a (MIR146A) is presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-146a (Homo sapiens microRNA 146A, MIR146A) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-146a. Based on this alignment, a structural model for miRNA-146a was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-155\u003c/h2\u003e\n\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-155 (NCBI Reference Sequence: NR_030784.1) was retrieved from the GenBank database in FASTA format. This sequence encodes a 65-nucleotide linear non-coding RNA (ncRNA). All FASTA-formatted sequences were obtained with annotations from NCBI - Graphics. The analysis for Homo sapiens microRNA 155 (MIR155) is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe structural template for miRNA-155 was constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 155) and the template structure. Assessment tools were employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 155 was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-132\u003c/h2\u003e\n\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-132 (NCBI accession: NR_029674.1) was retrieved from GenBank in FASTA format. This sequence encodes a 101-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 132 (MIR132) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-132 (Homo sapiens microRNA 132, MIR132) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-132. Based on this alignment, a structural model for miRNA-132 was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-191\u003c/h2\u003e\n\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eAn inconsistency exists in the provided information for miRNA-191. The NCBI accession code for miRNA-132 (NR_029690.1) is used here, while the figure caption likely refers to a different miRNA. Please verify and correct the nucleotide sequence and NCBI accession code for miRNA-191. The analysis for the corrected miRNA-191 will be presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eFollowing the acquisition of the correct nucleotide sequence for miRNA-191, the structural template will be constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 191) and the template structure. Assessment tools will be employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 191 will be generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-21\u003c/h2\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-21 (NCBI accession: NR_029493.1) was retrieved from GenBank in FASTA format. This sequence encodes a 72-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 21 (MIR21) is demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-21 (Homo sapiens microRNA 21, MIR21) was acquired employing FASTA format; modeling was performed employing the RNAComposer, optimized and adjusted for alignment between structural templates and miRNA-21 nucleotide. Based on sequence alignment between the template structure and miRNA-21 nucleotide, a structural model was built for the nucleotide in question. So, employing the RNAComposer of comparative nucleotide modeling, we generated a homology model of microRNA 21 (MIR21), demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n\u003ch2\u003eMiRNA-203a\u003c/h2\u003e\n\u003cdiv id=\"Sec26\" class=\"Section4\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-203a (NCBI accession: NR_029620.1) was retrieved from GenBank in FASTA format. This sequence encodes a 110-bp linear ncRNA. The analysis for Homo sapiens microRNA 203a (MIR203A) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-203a (Homo sapiens microRNA 203a, MIR203A) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-203a. Based on this alignment, a structural model for miRNA-203a was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-203b\u003c/h2\u003e\n\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-203b (NCBI accession: NR_039859.1) was retrieved from GenBank in FASTA format. This sequence encodes an 86-nucleotide linear ncRNA. The analysis for Homo sapiens microRNA 203b (MIR203B) is demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-203b (Homo sapiens microRNA 203b, MIR203B) was obtained in FASTA format and used for modeling with the RNAComposer. The server optimized and adjusted the sequence for alignment with structural templates closely resembling miRNA-203b. Based on this alignment, a structural model for miRNA-203b was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNA-210\u003c/h2\u003e\n\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n\u003ch2\u003eNucleotide Sequence\u003c/h2\u003e\n\u003cp\u003eThe nucleotide sequence for miRNA-210 (NCBI accession: NR_029623.1) was retrieved from GenBank in FASTA format. This sequence encodes a 110-bp linear ncRNA. The analysis for Homo sapiens microRNA 210 (MIR210) is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec33\" class=\"Section4\"\u003e\n\u003ch2\u003eMolecular Model\u003c/h2\u003e\n\u003cp\u003eThe structural template for miRNA-210 was constructed based on the sequence alignment between its nucleotide sequence (Homo sapiens microRNA 210) and the template structure. Assessment tools were employed to evaluate the reliability of the designed structure. Subsequently, a homology model of Homo sapiens microRNA 210 was generated using comparative nucleotide modeling on the RNAComposer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003emiRNA are small, non-coding RNA that play critical roles in various physiological and pathophysiological processes. Over the past decade, our understanding of miRNA structure and function has advanced significantly. This study presents a tutorial on \u003cem\u003ein silico\u003c/em\u003e modeling of miRNA structures and demonstrates the predicted 3D structures of eight miRNAs known to be overexpressed in DFUs.\u003c/p\u003e \u003cp\u003eDFUs are a severe complication of diabetes mellitus, contributing significantly to morbidity and mortality. Understanding the underlying genetic and molecular mechanisms is crucial for improving treatment efficacy. Various miRNA and their target genes are involved in both tissue fibrosis and angiogenesis in DFUs.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eLocated on chromosome 5q33.3, miRNA-146a is implicated in the pathogenesis of many diseases by inhibiting target gene expression. The miRNA-146a family (including miRNA-146a and miRNA-146b) negatively regulates inflammatory gene expression in monocytes, endothelial cells, and epithelial cells. Additionally, miRNA-146a induces the innate immune response upon encountering antigens.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e While miRNA-146a expression is significantly reduced in DFUs themselves, studies show increased expression in surrounding tissues, suggesting context-dependent regulation.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Interestingly, treatment with a miR-146a inhibitor promotes faster wound healing in DFUs, potentially through increased vascular endothelial growth factor and fibronectin production.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe post-genomic era has witnessed significant technological advancements, leading to the expansion of microarrays and databases. However, a major challenge for researchers lies in extracting valuable information from this vast resource. Bioinformatics offers a multitude of tools for predicting miRNA structures, which can ultimately lead to a deeper understanding of miRNA transcriptional patterning. In this study, we investigated the sequence of miRNA-146a using the NCBI database, specifically the GenBank resource identified by the corresponding NCBI identifier. We then employed bioinformatics tools to analyze the encoded nucleotides within miRNA-146a and subsequently modeled its structure. Bioinformatics tools for miRNA prediction have become increasingly accessible due to the relative difficulty of performing experimental miRNA structure determination. \u003cem\u003eIn silico\u003c/em\u003e evaluation of miRNAs primarily relies on the analysis of their primary and secondary structures. Our literature review identified no prior studies presenting a 3D structure model for miRNA-146a. To address this gap, we employed the GenBank nucleotide database to retrieve the miRNA-146a sequence and subsequently utilized the RNAComposer online program to generate its predicted 3D model.\u003c/p\u003e \u003cp\u003eMiRNA-155 is a small, single-stranded, non-coding RNA located at chromosome 21q21.3 in humans. Previously known as B-cell Integration Cluster, it is encoded by the MIR155 host gene (MIR155HG). MiRNA-155 plays a diverse role in various physiological and pathological processes. Studies suggest its potential involvement in the pathogenesis of diabetic complications.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Treating chronic DFUs presents a significant challenge due to the conflicting requirements of managing inflammation and promoting re-epithelialization. Both processes can contribute to increased bacterial colonization, ultimately delaying wound healing. Research has shown an association between miR-155 and increased severity of DFUs.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Diabetic patients often exhibit reduced levels of miRNA-155 expression in peripheral blood mononuclear cells. Conversely, inhibition of miRNA-155 leads to decreased inflammation, possibly due to increased M2 macrophage polarization and type I collagen deposition.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Additionally, in immune cells, miRNA-155 plays a pro-inflammatory role by suppressing cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA literature review identified only one prior study presenting a 3D structural model of miRNA-155.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In this study, we employed a computational approach to analyze the miRNA-155 sequence and predict its 3D structure. We retrieved the sequence using the FASTA format from a nucleotide database. Subsequently, the RNAComposer program was utilized for 3D modeling. The program was optimized to achieve alignment between structural templates and the miRNA-155 sequence. This approach, based on structural homology, facilitated the generation of the predicted 3D model of miRNA-155. The development of accurate molecular models holds promise for advancing the development of novel therapeutic drugs that target specific organs.\u003c/p\u003e \u003cp\u003emiRNA-132 is a microRNA transcribed from an intergenic region on human chromosome 17 at location 17p13.3.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e It targets various factors involved in angiogenesis and inflammation, both of which are crucial processes in DFUs. miRNA-132 can promote endothelial cell proliferation and contribute to neovascularization through angiogenic factors.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Conversely, miRNA-132 can also modulate inflammation, potentially contributing to the underlying inflammation associated with diabetic insulin resistance.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Studies have shown the efficacy of miRNA-132 in promoting wound healing in diabetic mouse models, and a case study suggests its potential for topical application in human diabetic wounds.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Our literature review did not identify any published studies presenting a 3D structural model of miRNA-132. Therefore, we utilized a homology modeling approach based on the miRNA-132 sequence retrieved from the NCBI Nucleotide database. The RNAComposer web server was employed to generate a predicted 3D model of miRNA-132 using a high-homology structure as a template.\u003c/p\u003e \u003cp\u003emiRNA-191, a member of the miR-191 family, is located on human chromosome 3 at position 3p21.31.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Its expression modulates cell proliferation, apoptosis, and the cell cycle in various diseases. Additionally, miRNA-191 is a stress-sensitive miRNA, and low levels have been linked to diabetes mellitus.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e A recent study demonstrated altered plasma miRNA-191 profiles in diabetic patients with impaired wound healing compared to diabetic patients without chronic foot ulcers, suggesting a potential role in wound healing.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Our literature review identified no published secondary structure models for miRNA-191. Therefore, we employed a computational homology modeling approach to predict the 3D structure of Homo sapiens miRNA-191. The FASTA sequence of miRNA-191 was used in conjunction with a high-homology structure as a template on the RNAComposer web server to generate the predicted 3D model.\u003c/p\u003e \u003cp\u003eLocated within a coding gene for vacuole membrane protein on chromosome 17q23.2, human miRNA-21 plays a critical role in various diseases, biological development, and regulation of immunological processes.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Studies have shown that miR-21 expression is downregulated during diabetic wound healing. MiRNA-21 is crucial for fibroblast migration, collagen deposition, and exhibits pro-proliferative, anti-apoptotic, and anti-inflammatory properties.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Its expression varies throughout the diabetic wound healing process, with high levels in the inflammatory phase, decreasing during the proliferative phase, and significantly increasing during the remodeling phase.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Our literature review identified only one published study presenting a 3D structural model of miRNA-21.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Therefore, we employed the FASTA sequence of miRNA-21 retrieved from the GenBank nucleotide database to generate a predicted 3D model using the RNAComposer web server.\u003c/p\u003e \u003cp\u003emiRNA-203a and 203b, located on human chromosome 14q32.33, play a role in modulating the inflammatory response and cell proliferation, particularly important for skin development.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Studies have shown increased expression levels of miRNA-203 in DFUs. Notably, miRNA-203 is highly abundant in the epidermis and may play a significant role in diabetic wound healing. However, research suggests a positive correlation between miRNA-203 expression and diabetic foot ulcer severity.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Our literature review identified no published studies presenting 3D structural models for either miRNA-203a or miRNA-203b. Therefore, we utilized the GenBank nucleotide database to retrieve the sequences of both miRNAs and subsequently employed the RNAComposer web server to generate predicted 3D models for miRNA-203a and miRNA-203b.\u003c/p\u003e \u003cp\u003emiRNA-210, located on chromosome 11p15.5, is involved in various biological processes within the human body, regulating cellular functions such as metabolism, cell proliferation, apoptosis, and angiogenesis.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Studies have shown reduced expression of miR-210 in DFUs. Local reconstitution using miR-210 mimics significantly improves diabetic wound healing, potentially by decreasing the wound's oxygen consumption rate through a local reduction of reactive oxygen species (ROS) levels in the wound tissue.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Our literature review did not identify any published studies presenting a secondary structure model for miRNA-210. Therefore, we employed a computational approach to build a predicted 3D structure model for Homo sapiens miRNA-210. The FASTA sequence of miRNA-210 was used in conjunction with a high-homology structure as a template on the RNAComposer web server to generate the model.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003emiRNAs are small non-coding RNAs with essential functions in various biological processes. Their structure and function are determined by their nucleotide sequence. Computational structure prediction methods can be employed to identify potential binding sites within these nucleotides, which have significant value for clinical and pharmacological applications. This study used \u003cem\u003ein silico\u003c/em\u003e approaches to analyze the secondary structures of eight selected miRNAs, known to be overexpressed in DFUs, and modulation of the expression of these miRNAs may be a promising strategy for the treatment of DFUs.\u003c/p\u003e"},{"header":"DECLARATIONS","content":" \u003ch2\u003eDisclosure of potential conflicts of interest:\u003c/h2\u003e \u003cp\u003eNone of the authors have any potential conflicts of interest to disclose.\u003c/p\u003e "},{"header":"REFERENCES","content":"\u003col\u003e\n\u003cli\u003eZhang P, Lu J, Jing Y, Tang S, Zhu D, Bi Y. Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis. Ann Med. 2017;49(2):106-116.\u003c/li\u003e\n\u003cli\u003eBrocco E, Ninkovic S, Marin M, et al. Diabetic foot management: multidisciplinary approach for advanced lesion rescue. J Cardiovasc Surg (Torino). 2018;59(5):670-684.\u003c/li\u003e\n\u003cli\u003eBandyk DF. The diabetic foot: Pathophysiology, evaluation, and treatment. Semin Vasc Surg. 2018;31(2-4):43-48.\u003c/li\u003e\n\u003cli\u003eMoura J, S\u0026oslash;rensen A, Leal EC, et al. microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation. Sci Rep. 2019;9(1):5836.\u003c/li\u003e\n\u003cli\u003eSaliminejad K, Khorram Khorshid HR, Soleymani Fard S, Ghaffari SH. Na overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol. 2019;234(5):5451-5465.\u003c/li\u003e\n\u003cli\u003eLiang L, Stone RC, Stojadinovic O, et al. Integrative Analysis of miRNA and mRNA Paired Expression Profiling of Primary Fibroblast Derived from Diabetic Foot Ulcers Reveals Multiple Impaired Cellular Functions. Wound Repair Regen. 2016;24(6):943-953.\u003c/li\u003e\n\u003cli\u003eMeng S, Cao J, Zhang X, et al. Downregulation of microRNA-130a contributes to endothelial progenitor cell dysfunction in diabetic patients via its target Runx3. PLoS One. 2013;8(7):e68611.\u003c/li\u003e\n\u003cli\u003eWang H, Li X, Li T, et al. Oncol Lett. 2019;18(5):5033-5042.\u003c/li\u003e\n\u003cli\u003eBruhn-Olszewska B, Korzon-Burakowska A, Gabig-Cimińska M, Olszewski P, Węgrzyn A, Jak\u0026oacute;bkiewicz-Banecka J. Molecular factors involved in the development of diabetic foot syndrome. Acta Biochim Pol. 2012;59(4):507-13.\u003c/li\u003e\n\u003cli\u003eFeng B, Chen S, Zhang L, Cao Y, Chakrabarti S. miRNA-146a and miRNA200b Antagomirs Accelerate Wound Healing through the Regulation of VEGF and Fibronectin. Journal of Pharmacy and Pharmacology. 2014;2:104-113.\u003c/li\u003e\n\u003cli\u003eFaraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: a typical multifunctional microRNA. Biochim Biophys Acta. 2009;1792(6):497-505.\u003c/li\u003e\n\u003cli\u003eKhamaneh AM, Alipour MR, Sheikhzadeh Hesari F, Ghadiri Soufi F. A signature of microRNA-155 in the pathogenesis of diabetic complications. J Physiol Biochem. 2015;71(2):301-9.\u003c/li\u003e\n\u003cli\u003eGuo Y, Lin C, Xu P, et al. AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes. Sci Rep. 2016;6:36416.\u003c/li\u003e\n\u003cli\u003eZhang Y, Sun E, Li X, et al. miR-155 contributes to Df1-induced asthma by increasing the proliferative response of Th cells via CTLA-4 downregulation. Cell. Immunol. 2017;314:1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eRenrick AN, Thounaojam MC, de Aquino MTP, et al. Bortezomib Sustains T Cell Function by Inducing miR-155-Mediated Downregulation of SOCS1 and SHIP1. Front Immunol. 2021;12:607044.\u003c/li\u003e\n\u003cli\u003eSalta E, De Strooper B. microRNA-132: a key noncoding RNA operating in the cellular phase of Alzheimer\u0026apos;s disease. FASEB J. 2017;31(2):424-433. \u003c/li\u003e\n\u003cli\u003eGougelet A, Pissaloux D, Besse A, et al. Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response. Int J Cancer. 2011;129(3):680-90. \u003c/li\u003e\n\u003cli\u003eStrum JC, Johnson JH, Ward J, et al. MicroRNA 132 regulates nutritional stressinduced chemokine production through repression of SirT1. Mol Endocrinol. 2009;23(11):1876-84.\u003c/li\u003e\n\u003cli\u003eLi X, Li D, Wang A, et al. MicroRNA-132 with Therapeutic Potential in Chronic Wounds. J Invest Dermatol. 2017;137(12):2630-2638.\u003c/li\u003e\n\u003cli\u003eMIR191 microRNA 191 [Homo sapiens (human)]. Available from: https://www.ncbi.nlm.nih.gov/gene/406966 (accessed March 24, 2024).\u003c/li\u003e\n\u003cli\u003eNagpal N, Kulshreshtha R. miR-191: an emerging player in disease biology. Front Genet. 2014;5:99.\u003c/li\u003e\n\u003cli\u003eDangwal S, Stratmann B, Bang C, et al. Impairment of Wound Healing in Patients With Type 2 Diabetes Mellitus Influences Circulating MicroRNA Patterns via Inflammatory Cytokines. Arterioscler Thromb Vasc Biol. 2015;35(6):1480-8.\u003c/li\u003e\n\u003cli\u003eKumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and disease. RNA Biol. 2011;8(5):706-13.\u003c/li\u003e\n\u003cli\u003eTana C, Giamberardino MA, Cipollone F. microRNA profiling in atherosclerosis, diabetes, and migraine. Ann Med. 2017;49(2):93-105.\u003c/li\u003e\n\u003cli\u003eShang YY, Fang NN, Wang F, et al. MicroRNA-21, induced by high glucose,modulates macrophage apoptosis via programmed cell death 4. Mol Med Rep. 2015;12(1):463-9.\u003c/li\u003e\n\u003cli\u003eBaisden JT, Boyer JA, Zhao B, Hammond SM, Zhang Q. Visualizing a protonated RNA state that modulates microRNA-21 maturation. Nat Chem Biol. 2021;17(1):80-88. \u003c/li\u003e\n\u003cli\u003eZang J, Hui L, Yang N, Yang B, Jiang X. Downregulation of MiR-203a Disinhibits Bmi1 and Promotes Growth and Proliferation of Keratinocytes in Cholesteatoma. Int J Med Sci. 2018;15(5):447-455.\u003c/li\u003e\n\u003cli\u003eLiu J, Xu Y, Shu B, et al. Quantification of the differential expression levels of microRNA-203 in different degrees of diabetic foot. Int J Clin Exp Pathol. 2015;8(10):13416-20.\u003c/li\u003e\n\u003cli\u003eBavelloni A, Ramazzotti G, Poli A, et al. MiRNA-210: A Current Overview. Anticancer Res. 2017;37(12):6511-6521.\u003c/li\u003e\n\u003cli\u003eModaghegh MHS, Saberianpour S, Amoueian S, Shahri JJ, Rahimi H. The effect of redox signaling on extracellular matrix changes in diabetic wounds leading to amputation. Biochem Biophys Rep. 2021;26:101025.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic foot, microRNA, Molecular structure","lastPublishedDoi":"10.21203/rs.3.rs-4278543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4278543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntrodução: \u003c/strong\u003eDiabetic neuropathy-associated vasculopathy is a significant risk factor for the development of diabetic foot ulcers (DFUs). In the context of DFUs, miRNAs can influence the cascade of molecular events that culminate in healing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: To design \u003cem\u003ein silico\u003c/em\u003e the molecular structures of microRNAs (miRNAs) overexpressed in diabetic foot ulcer healing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We conducted a search for the nucleotide sequences of eight miRNAs overexpressed in DFUs, and the following miRNAs were selected: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. These miRNAs were selected for evaluation in this study based on pre-clinical evidence, differential expression in DFUs, and therapeutic potential. Subsequently, the molecular structures of the eight miRNAs were designed \u003cem\u003ein silico\u003c/em\u003e. The nucleotide sequences were retrieved from GenBank, the genetic sequence database of the National Center for Biotechnology Information. The obtained sequences were aligned using multiple alignment algorithms from the RNA Fold web server. RNAComposer, an automated miRNA structure modeling server, was employed for the \u003cem\u003ein silico\u003c/em\u003e modeling of the structures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We performed a search for the nucleotide sequences and designed the molecular structures of the following miRNAs overexpressed in diabetic foot ulcer healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. We generated a tutorial on the molecular models of these eight miRNAs overexpressed in the diabetic foot, based on \u003cem\u003ein silico\u003c/em\u003e projections of their molecular structures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study demonstrates the \u003cem\u003ein silico \u003c/em\u003edesign of secondary structures for a selection of eight miRNAs overexpressed in diabetic foot ulcer healing, utilizing techniques from computational biology.\u003c/p\u003e","manuscriptTitle":"Diabetic Foot: A MicroRNA-Centric Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 07:18:29","doi":"10.21203/rs.3.rs-4278543/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab6e42e9-7d0a-4b1a-b6b5-00f76d505028","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30772913,"name":"Endocrinology \u0026 Metabolism"},{"id":30772914,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2024-04-18T07:18:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-18 07:18:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4278543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4278543","identity":"rs-4278543","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}