Endotipizacija kroničnega rinosinuzitisa s sekvenciranjem RNA

Urbančič, Jure (avtor), Hočevar Boltežar, Irena (mentor), Rijavec, Matija (komentor)

Izhodišča: Kronični rinosinuzitis (KRS) je bolezen sluznice obnosnih votlin z dvema fenotipoma, z nosno polipozo (KRSzNP) in brez (KRSbNP). Fenotipska delitev pa ne pojasni variabilnosti bolezni, porazdelitve endotipov ali odziva na terapijo. Poznavanje vnetnih mehanizmov, njihova identifikacija s pomočjo analize transkriptoma, lahko pojasni razvoj bolezni, njen naravni potek in odziv na terapijo. Metode: Raziskava je bila zasnovana kot kohortno-prospektivna bolnikov s primarnim KRS. Kontrolno skupino so sestavljali bolniki z zdravo nosno sluznico. Sekvenciranje RNA (RNAseq) je bilo izvedeno na platformi Illumina NovaSeq6000. Pridobljene rezultate smo nato analizirali in interpretirali, in sicer smo rezultate prilegali na referenčni genom, filtrirali in izračunali stopnjo izražanja za posamezen vzorec. Izvedli smo karakterizacijo diferencialno izraženih genov ter funkcionalne analize, ki so vključevale bolezni in funkcije, kanonične poti in transkripcijske regulatorje. Rezultati: Analiza transkriptoma je bila opravljena na vzorcih 22 oseb s KRSzNP, 11 s KRSbNP in 15 kontrolah. V tkivu nosne sluznice smo ugotovili visoko korelacijo med fenotipoma (r=0,637, P<0,001). Vendar smo opazili tudi razlike, saj je bilo ugotovljenih 375 diferencialno izraženih genov (DEG), specifičnih za KRSzNP, in 328 DEG, specifičnih za KRSbNP. Poleg tega smo identificirali tudi 75 DEG, specifičnih za posamezen fenotip. Funkcionalna analiza je pokazala na imunsko regulacijo, celično gibanje in vnetje, kot najbolj značilne poti pri KRS. Zaključki: S pomočjo analize celotnega transkriptoma smo odkrili razlike med fenotipi pri imunski regulaciji KRSzNP prek NF-kß, signalizacije preko Tollu podobnega receptorja in signalizaciji, regulaciji HIF1?, poti tipa 2 (T2), poteh IL-6 in IL-15 ter rekrutiranju in regulaciji različnih imunskih celic. Pri KRSbNP je visoko aktivirana regulacija imunskega odziva NFAT in s kalcijem inducirana apoptoza celic T. Potrdili smo tudi visoko aktivacijo regulatorjev, ki sprožijo vnetne procese, uravnavajo imunske poti, znane molekule prirojenega imunskega odziva, za katere se je že predhodno izkazalo, da sodelujejo pri KRS, kot so bakterijski produkti in regulatorji celic T.

• Jezik: Slovenski jezik
• Ključne besede: kronični rinosinuzitis, tranksriptom, RNA, RNA sekvenciranje, genetika
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2023
• PID: 20.500.12556/RUL-152818
• Datum objave v RUL: 08.12.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Endotyping of chronic rhinosinusitis using RNA sequencing

Izvleček

Background: Chronic rhinosinusitis (CRS) is a disease of the sinuses with two phenotypes, with nasal polyposis (CRSwNP) and without (CRSsNP). Phenotypes do not explain the variability of the disease, the global distribution of its endotypes, or the response to therapy. Knowledge of different inflammatory mechanisms obtained through transcriptome can help explain the development of the disease. Methods: The study was designed as a prospective cohort study of patients with an apparent primary CRS. The control group consisted of patients with healthy nasal mucosa. RNA was extracted and assessed for purity and integrity. RNA sequencing (RNAseq) was performed on an Illumina NovaSeq6000 platform. The analysis included the removal of low-quality reads, adapter removal, alignment with the reference sequence, and normalization of reads. Functional analyses included the identification of diseases and functions, canonical pathways, and transcriptional regulators. Results: Transcriptome analysis was performed on samples from 22 subjects with CRSwNP, 11 with CRSsNP, and 15 controls. In tissue samples, we found a high correlation between CRSwNP and CRSsNP (r = 0.637, P < 0.001). However, differences were also observed, with 375 CRSwNP-specific differentially expressed genes (DEGs) and 328 CRSsNP-specific DEGs identified. In addition, we also identified 75 DEGs specific to each phenotype. Enrichment of DEGs shows immune regulation, overall movement, and inflammation as the best-characterized pathway in CRS. Conclusions: Analysis revealed differences between phenotypes in CRSwNP immune regulation via NF-kβ, Toll-like receptor signaling and signaling, HIF1α regulation, T2-pathway, IL-6 and IL-15 pathways, and recruitment and regulation of various immune cells. NFAT immune response regulation and calcium-induced T cell apoptosis are highly activated in CRSsNP. As well as, high activation of regulators that trigger inflammatory processes regulate immune pathways and known molecules of the innate immune response, which have previously been shown to be involved in KRS, such as bacterial products and T-cell regulators.

• Ključne besede: Chronic rhinosinusitis, transcriptome, RN, RNA sequencing