Izražanje izbranih nekodirajočih RNA pri bolnikih z amiotrofično lateralno sklerozo : doktorska disertacija

Dolinar, Ana (author), Ravnik Glavač, Metka (mentor)

Amiotrofična lateralna skleroza (ALS) je nevrodegenerativna bolezen, ki prizadene zgornje in spodnje motorične nevrone. Pri slabih 30 % bolnikov najdemo mutacije v enem izmed 33 bolezenskih genov, nastanek bolezni pri ostalih bolnikih pa je verjetno povezan s kombinacijo različnih epigenetskih dejavnikov. V okviru doktorske disertacije smo preučevali izražanje krožnih RNA (circRNA), mikro RNA (miRNA), njihovih gostiteljskih genov in metilacijo DNA v promotorskih regijah intergenskih miRNA. Z uporabo mikromrež in kasnejšim potrjevanjem teh rezultatov smo odkrili šest zvišano izraženih (hsa_circ_0000567, hsa_circ_0005218, hsa_circ_0035796, hsa_circ_0043138, hsa_circ_0063411 in hsa_circ_0088036) ter eno znižano izraženo (hsa_circ_0023919) circRNA v levkocitih bolnikov z ALS. Hsa_circ_0023919, hsa_circ_0063411 in hsa_circ_0088036 so se izkazale kot najbolj obetavni biološki označevalci. V izražanju treh gostiteljskih genov, PICALM (hsa_circ_0023919), SUSD1 (hsa_circ_0088036) in AATK (miR-657), ni bilo razlik med bolniki in zdravimi kontrolami, TNRC6B (hsa_circ_0063411) pa je bil pri bolnikih zvišano izražen. Metilacija preučevanih mest CpG v promotorskih regijah intergenskih miRNA miR-182, miR-217 in miR-451a ne vpliva na njihovo izražanje. MiR-182 in miR-451a sta bili pri bolnikih zvišano izraženi, stopnje izražanja miR-217 in miR-657 pa zaradi nespecifičnih pomnožkov nismo mogli določiti. Izražanje circRNA je večinoma šibko ali zmerno negativno povezano z različnimi kliničnimi značilnostmi bolnikov ter zmerno do močno pozitivno povezano z izražanjem drugih circRNA. Opazili smo šibko negativno povezavo med izražanjem miR-182 in izražanjem njenega predpostavljenega tarčnega gena TNRC6B, med izražanjem miRNA in njihovih predpostavljenih tarčnih circRNA pa nismo opazili povezav.

• Language: Slovenian
• Keywords: nevrodegenerativne bolezni, amiotrofična lateralna skleroza, epigenetika, nekodirajoče RNA, circRNA, miRNA, metilacija
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-121950
• COBISS.SI-ID: 36968195
• Publication date in RUL: 11.11.2020

Secondary language

• Language: English
• Title: Expression of selected non-coding RNAs in patients with amyotrophic lateral sclerosis : doctoral dissertation

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons. Approximately 30% of patients have a mutation in one of 33 disease-causing genes, while disease development in others is probably due to the combination of various epigenetic factors. In this thesis, we analysed the expression of circular RNAs (circRNAs), micro RNAs (miRNAs), their host genes, and the methylation levels of selected CpG sites in promoter regions of intergenic miRNAs. Using microarrays and subsequent validation of its results, we discovered six upregulated (hsa_circ_0000567, hsa_circ_0005218, hsa_circ_0035796, hsa_circ_0043138, hsa_circ_0063411, and hsa_circ_0088036) and one downregulated (hsa_circ_0023919) circRNA in leukocyte samples of ALS patients. Hsa_circ_0023919, hsa_circ_0063411, and hsa_circ_0088036 were identified as the most promising biomarkers. Expression levels of three host genes, PICALM (hsa_circ_0023919), SUSD1 (hsa_circ_0088036), and AATK (miR-657), showed no difference between patients and healthy controls, while TNRC6B (hsa_circ_0063411) was upregulated in patients. Methylation levels of selected CpG sites in promoter regions of intergenic miRNAs miR-182, miR-217, and miR-451a does not affect their expression levels. MiR-182 and miR-451a were upregulated and due to nonspecific qPCR products we were unable to determine miR-217 and miR-657 expression levels. CircRNA expression showed weak to moderate negative correlation with several clinical characteristics of ALS patients and moderate to strong correlation with expression levels of other circRNAs We observed a weak negative correlation between expression of miR-182 and expression of its predicted target gene TNRC6B, while no correlation was observed between the expression of miRNAs and their predicted target circRNAs.

• Keywords: neurodegenerative diseases, amyotrophic lateral sclerosis, epigenetics, non-coding RNAs, circRNA, miRNA, methylation