The Role of miRNA in oral lichen planus pathogenesis

Abstract

Oral lichen planus (OLP) is a chronic T-cell-mediated immune disease of unknown aetiology. Micro-RNA (miRNAs) are short non-coding RNAs capable of regulating mRNA that are closely correlated with cytokines in various inflammatory diseases. The aim of this study was to investigate the profile of miRNAs in a cohort of OLP patients and their interaction with potential target genes. Oral biopsy specimens were taken from a total of 32 patients: 24 patients, 12 females, , with an average age of 57.37 had been diagnosed with OLP whereas 8 patients, 5 females with an average age of 57.55 affected by oral disorders different from OLP causing white patches, were used as the control group. All experimental procedures were approved by the NRES Committee North East - Newcastle & North Tyneside (13/NE/0368) and the enrolled patients gave their informed written consent. Total RNA was extracted from fresh frozen biopsies and the paraffined tissues were used for immunohistochemical studies. The Nano string nCounter Analysis System was used to analyse total RNA samples and the raw data was processed using the nSolver software version 4.0. Nano String expression was confirmed by RT-qPCR analysis. Genes targeting up and down regulated miRNAs were identified using the following four tools: Target Scan human (www.targetscan.org), DIANA (www.diana.imis.athenainnovation.gr/DianaTools), Exiqon (www.exiqon.com/miRSearch) and mirTarBase (www.mirtarbase.mbc.nctu.edu.tw). Predicted target genes were further narrowed down to focus on genes that influence signalling pathways in OLP. RT-qPCR was employed to investigate the expression of target genes in OLP and controls. Non parametric statistical tests were employed to analyse the results. Probability values <0.05 were considered statistically significant. The results reveal that OLP is mainly a T-cell mediated disease characterized by overexpression of IFN-γ and TNF-α and apoptosis markers. NanoString analysis showed that the following 9 miRNAs were upregulated in OLP tissues: miR-155, miR-146a, miR 3195, miR-342, miR-4516, miR-21, miR-29a, miR-193, and miR-222. Contrarily, the following 9 miRNAs: miR-221, let-7, miR-23b, miR-200b, miR-149, miR-205, miR-23a, miR-27b, miR-95, were downregulated in OLP tissues. Two upregulated, namely miR 4516 and miR-222 and 2 downregulated miRNAs, miR-149, miR-95, were novel discoveries, whilst the remaining miRNAs were confirmation of previous findings. NanoString findings have been confirmed by RT-qPCR results for the following 5 upregulated miRNAs, miR-155, miR-146a, miR-29a, miR-222 and miR-342 and two downregulated miRNAs, namely miR-205 and miR23b. The expression of three target genes, namely MYC for miR-29a, interleukin-24 (IL-24) and MMP1 for miR-205 was found to have a negative correlation with the respective miRNA. This, suggests that MYC, IL-24 and MMP1 could be regulated by the above miRNAs in OLP. In conclusion, the work presented in this thesis suggests that miRNAs could be involved in both the immunopathogenesis and malignant transformation of OLP