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.