Determination of Changing Expression of miR-212 and EGFR Genes in Clinical Samples of Trichophyton rubrum Dermatophyte in Infected Sites compared to Adjacent Healthy

Abstract

Introduction & Goals:Skin infections with dermatophytes are common in the world and are called Tinea. As an anthropophilic fungus, Trichophyton rubrum is the most frequent cause of Tinea in the world. Antimicrobial peptides (AMPs) such as HBD-3 and RNase7 are effective molecules in innate immunity of the skin, which have potential antimicrobial effect,rapidly kill microorganismsand are affected by EGFR gene,the increasing expression of which in skin cells activates them and prevents the colonization of organisms, including dermatophytes in keratinocytes. However, through increasing expression of microRNAs, and in particular miR-212 in this study, the mRNA of EGFR gene is silenced and colonization of the dermatophyte in the skin leads to dermatophytosis. The aim of this study was to determine the changes in the expressions of miR-212 and EGFR genes in clinical specimens infected with Trichophyton rubrum dermatophyte in infected sites compared to adjacent healthy sites. Methods: Collection of clinical specimens was based on 36 samples of tissue infected with Trichophyton rubrum, collection of 36 control samples from the margin of the fungus-infected tissues in the same patients, extraction of whole RNA and its optimization, synthesis and optimization of cDNA, amplification of EGFR and miR-212 genes by Real-Time PCR (SYBER green), data collection and analysis was performed. Results: After culture of skin flakes on Mycosel agar medium and the appearance of Trichophyton rubrum colonies, a slide culture was prepared from the colonies to examine the reproductive organs of the fungus and the presence of T. rubrum was confirmed by 40x microscopy. After extracting whole cellular RNA from each of the clinical samples, the concentration of each sample was determined with Nanodrop device. The values obtained for the samples ranged 1.7-1.9 by the device, which indicate the acceptable purity of the extracted RNA. Subsequently, to determine the quality of RNA and maintain its integrity, the RNA extracted from each sample was run on 1% agarose gel. A gentle, uniform smear was thoroughlyexamined from top to bottom of the gel, and 28S, 18S, and 5S ribosomal RNA bands were clearly visible in each sample. Finally, Real-TimePCR was performed on both genes along with internal control genes (GAPDH and miR-103a-3p) and the resulting curves are shown in the fourth chapter. Conclusion: Bioinformatics analysis showed that miR-212 can affect EGFR as a potential target; however, functional studies are needed to demonstrate it. MiR-212 in tissue samples infected with Trichophyton rubrum dermatophyte significantly reduced the expression of EGFR gene, and the expression of miR-212 gene increased by about eight fold compared to that of EGFR gene. On the other hand, in tissue samples used as control, the expression of miR-212 was much less1 than that of EGFR gene, so the subject has been able to resist the invasion of T. rubrum in the peripheral areas of the lesion due to the presence of EGFR gene and consequent presence of AMPs.