Investigation of miR-96-5P and miR-5194 expression levels in nasal mucosa and polyp samples posisive for Helicobacter pylori CagA gene using molecular methods

Nasal polyps are semi-transparent, round, pale gray masses that arise as a result of chronic mucosal inflammation in the nasal and paranasal cavities. Helicobacter pylori, thought to be a cause of nasal polyps, carries the cagA gene, which induces severe inflammation in the body. Since inflammation plays a significant role in nasal polyps, it is believed that H.pylori infections may influence the inflammatory processes in the nasal area. The aim of this thesis is to investigate the presence of H.pylori cagA in nasal mucosa and polyps using molecular methods, and to clarify the critical role of this protein in the formation of nasal polyps in cagA-positive samples through specific miRNA biomarker analyses. This study is an important step in understanding the etiology of H.pylori infections and developing new treatment methods. In this study, the expression levels of miR-5194 and miR-96-5p were examined in nasal mucosa and polyp samples where H.pylori cagA was found positive by PCR method. For this purpose, tissue samples from 45 patients 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. One part of the tissue samples, divided into two under sterile conditions, was used for DNA extraction to determine the presence of H.pylori and cagA gene regions through PCR. RNA isolation was then performed on the remaining polyp samples from patients who showed positive results using Trizol. Quality control of RNA isolation was carried out on a 1% agarose gel containing 3.5 μL of RedSafe. Bands were visualized using the SYNGENE G-BOX ChemiXRQ UV gel documentation system, and the degradation status of the RNAs was checked. The quantity and yield of RNA molecules were measured using the Qubit HS RNA Assay Kit (Catalog No: Q32852, Q32855) with the Qubit Fluorometer device. cDNA synthesis was performed using ABScript III RT Master Mix for qPCR (RK20428) on RNA samples of appropriate quality. The cDNA samples were then subjected to PCR reactions in the Applied Biosystems Step One Plus Real-Time system. The expression levels of the genes in the tissue samples were determined. U6 was used as the housekeeping gene for normalization during Real-Time PCR. After cDNA synthesis, a qRT-PCR reaction was set up to determine the mRNA gene expression levels of miR-5194 and miR-96-5p. The reaction mixture and thermal cycling conditions were performed according to the manufacturer's protocol using the Genious 2X SYBR Green Fast qPCR Mix (High ROX Premixed) (RK21206). The results of the qRT-PCR were analyzed using the DataAssist v3.01 program to reveal differences between groups. The H.pylori PCR results obtained revealed the presence of H.pylori DNA and cagA gene regions in nasal polyps. In this study, changes in the expression levels of miR-96-5p and miR-5194 were investigated. It was observed that miR-96-5p remained at similar levels in the polyp group compared to the control group, showing no significant change. On the other hand, miR-5194 was found to be significantly elevated in the polyp samples and expressed at higher levels in polyp tissues Keywords: CagA gene, Helicobacter pylori, miRNA, Molecular, Nasal polyp