The investigation of the expression levels of miR-628-3p and miR-4455 in sinonasal mucosa and polyp samples positive for Aspergillus using molecular methods

Aspergillus species are microorganisms belonging to the class of mold fungi that can have significant effects on human health. The presence of Aspergillus species in sinonasal mucosa and polyp samples is particularly associated with the pathogenesis of diseases such as chronic sinusitis. The aim of the study was to demonstrate how Aspergillus species accumulate in sinus polyps and mucosal tissue, and how these fungi interact with the expression levels of miR-628-3p and miR-4455 in the development of disease using molecular methods. In this study, tissue samples from 45 patients who were diagnosed with nasal polyps at the Ear, Nose, and Throat Clinic of Turgut Özal Medical Faculty, İnönü University, Malatya, Turkey, between October 2024 and December 2024, were evaluated. The samples in the study were divided into two categories: mycological and molecular tests. Some samples were examined under a light microscope using 10% potassium hydroxide (KOH), while others were cultured on Sabouraud Dextrose Agar (Merk, Darmstadt, Germany) at 30°C for 14 days. For DNA extraction, a DNA extraction kit (Invetech, Berlin, Germany) was used according to the manufacturer's instructions. Afterward, the expression levels of miR-4455 and miR-628-3p were analyzed in Aspergillus-positive samples. RNA isolation was first performed using Trizol. The quality control of the RNA isolation was carried out using a 1% agarose gel containing 3.5 μL RedSafe, and the bands were visualized with the SYNGENE G-BOX ChemiXRQ UV gel imaging system to check for RNA degradation. The quantity and yield of RNA molecules were measured using the Qubit HS RNA Assay Kit (Catalog No: Q32852, Q32855) and a Qubit Fluorometer. cDNA synthesis was performed from the RNA samples using ABScript III RT Master Mix for qPCR (RK20428). Real-time PCR reactions were carried out with the synthesized cDNA samples on the Applied Biosystem Step One Plus Real-Time device. After cDNA synthesis, qRT-PCR reactions were set up to determine the expression levels of miR-4455 and miR-628-3p. The results obtained from QRT-PCR were analyzed using the DataAssist v3.01 software to identify differences between the groups. As a result, in Aspergillus-positive samples, miR-4455 showed higher expression compared to the control group, while miR-628-3p exhibited a lower fold change with a decrease in expression. Specifically, the expression levels of miR-628-3p in control and polyp samples were found to be lower compared to miR-4455. The increased expression of miR-4455 suggests that this miRNA may contribute to polyp formation or play an important role in inflammatory processes. Similarly, miR-628-3p may be involved in this process with its expression, but its effect may be more limited compared to miR-4455.