Investigation of the Role of Micro-RNAs in the Immunopatho-genesis of Oral Lichen Planus Patients

Description

Background and Objectives: Lichen planus (LP) is a chronic inflammatory disorder affecting the skin, mucous membranes, and other tissues. Its exact etiology remains elusive, but the immunopathogenesis of LP has gained significant attention due to its complex interplay between immune dysregulation and tissue response. We aim to investigate the importance of understanding LP immunopathogenesis, explicitly focusing on the role of microRNAs (miRNAs) in modulating the disease process. Materials and Methods: In our research, punch biopsy samples were collected from 85 patients, including 52 Oral Lichen Planus (OLP) and 33 healthy volunteer patients, according to specific inclusion and exclusion criteria. Samples were analyzed for miR-21, p53, and p63 expression using real-time Polymerase Chain Reaction (RT/PCR). Results: The OLP group was compared to a control group, revealing increased miR-21 and p53 expression. Further comparison of the OLP group’s miR-21, p53, and p63 expression showed a weak positive correlation between p53 and miR-21 and a weak negative correlation between p63 and miR-21. These findings suggest that miR-21 and its potential targets, p53 and p63, play crucial roles in the pathogenesis and malignant potential of OLP. Conclusions: In conclusion, our research suggests that miR-21 and its target genes p53 and p63 are crucial in the molecular pathogenesis and cancerous potential of OLP. This understanding can lead to targeted treatment approaches for this pre-cancerous condition.

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In our preliminary analysis, miRwalk (Ruprecht-Karls-Universität Heidelberg, Medizinische Fakultät Mannheim, Germany), an internet-based, academic bioinformatics tool, was used to examine miR-21’s ability to target promoter, 5′UTR, intragenic and 3′UTR regions of genes and approximately 2000 genes were identified accordingly. These genes were then filtered according to their expression in epithelial cells in online expression databases. In addition, the validated target p53 and p63 genes targeted by mir-21 were identified by searching the databases of previous studies. Total RNA isolation was performed using the Qiagen miRNeasy mini kit (Qiagen LLC, Germantown, MD, USA) protocol. miRNeasy mini kit has stood out in many studies with its capacity to isolate ~18 nt long RNA molecules with high specificity and concentration. After isolating total RNA from the patient and control tissues, a spectrophotometer (nd-1000, NanoDrop Technologies, Inc., Wilmington, NC, USA) and a Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) were used to determine RNA concentration and quality. Determining the concentration and quality of RNA in the extracted samples is critical before PCR. For this purpose, miRNA concentration and the ratio of miRNA to small RNA in extracted total RNA were determined using an Agilent 2100 Bioanalyzer. OD measurements of RNAs were diluted to 100ng/ μll. Random hexamer cDNA synthesis was performed for p53 and p63 genes. cDNA synthesis was performed using an S24 gene stem-loop RT primer to be studied as a housekeeping gene. This protocol was performed with Thermoscientific’s cDNA synthesis kit. After target cDNA synthesis, miR-21 gene expression level was determined by quantitative real-time PCR method using primers explicitly designed for miR-21 and UPL probe number 21. Since the sample quality was insufficient, two samples from the OLP patient group (H32, H50) and three from the healthy control group (K8, K23, K27) were excluded. The p53 gene expression level was determined by quantitative real-time PCR using primers explicitly designed for p53 and UPL probe number 73. One sample (K27) was excluded from the control group due to insufficient sample quality. The p63 gene expression level was determined by quantitative real-time PCR using primers explicitly designed for p63 and UPL probe number 42. Since the sample quality was insufficient, two samples (K1, K27) were excluded from the control group. To be studied as a housekeeping gene, the expression level of the S24 gene was determined by quantitative real-time PCR method using a unique primer and probe. Real-time quantitative PCR is a method that allows real-time observation of the amount of amplification occurring during the reaction by using fluorescent dyes.

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