Mitokondri Hasarı ile İlişkili miR-382-5p ve miR-409-3p’nin Hedef Genlerinin Araştırılması
Özet
Mitochondrial dysfunction and dysmorphology in skeletal muscle are observed as a common and secondary finding in most neuromuscular diseases that have different clinic, genetic and pathologic background, other than mitochondrial cytopathies. In our previous study (HUBAP Project no: TSA-2017-14413), common miRNAs associated with secondary mitochondrial damage observed in the ethiopathogenesis of neuromuscular diseases, in which different nuclear genes are responsible, were identified for the first time in the literature. Cell based functional analyses revealed that increased expression of miR-382-5p and miR-409-3p in skeletal muscle cells both reduce ATP synthesis and disrupt organelle morphology, thus these miRNAs were identified as priority candidates. In this thesis study, it was aimed to identify candidate mitochondrial target genes of miR-382-5p and miR-409-3p, which we associated with secondary mitochondrial damage observed in skeletal muscle. For this purpose, in the first step, potential target genes were identified by bioinformatics analysis and overlapped with MitoCarta3.0 gene list to create a potential mitochondrial target gene list for each miRNA. Gene enrichment analyses using the Gene Ontology database identified priority candidate genes for both miRNAs involved in pathways related to cellular ATP production and/or mitochondrial morphology. After transfection of miR-382-5p and miR-409-3p miRNA mimics into C2C12 mouse myoblast cells, mRNA expression levels of Opa1, Uqcc1 and Mief1 for miR-382-5p and, Mfn1 and Uqcc1 for miR-409-3p, were analyzed by RT-qPCR at 48, 72 and 96 hours. It was concluded that increased miR-382-5p expression decreased the Mief1 mRNA level (2,29-fold, p=0,0286) 48 hours after transfection, and increased miR-409-3p expression decreased the Mfn1 level (2,11-fold, p=0,007937) 96 hours after transfection, at a statistically significant level compared to mimic negative control transfected cells. In the subsequent Western Blot analyses, no significant decrease in the Mief1 protein level could be detected in miR-382-5p transfected cells, whereas the amount of Mfn1 protein was found to be statistically significantly reduced compared to control cells at 96 hours after transfection (1,67-fold, p=0,0317), consistent with the RT-qPCR results.
