PhD Scientific Days 2022

Neurosciences II.

Investigation of the function of DNM2 gene in mitochondrial dynamics using siRNA gene silencing model

Text of the abstract

Background: The Dynamin2 protein (DNM2) has diverse roles in cell functions. DNM2 is a large GTPase involved in membrane trafficking and endocytosis. It has roles in clathrin mediated endocytosis at the plasma membrane and together with DRP1, in mitochondrial division. In this study, we tested the effect of DNM2 depletion on HeLa cells after siRNA silencing. DNM2 depletion blocks mitochondrial division and results in an elongated, hyper-fused mitochondrial network.
Methods: The silencing was performed for 3 days. The efficiency of gene silencing was analysed by real-time PCR and Western blotting. RNA sequencing was performed on the properly transfected samples on Illumina NextSeq platform. Bioinformatics analysis was performed focusing on changes of the mRNA expression.
Results: Gene silencing of siRNA was performed with 3 different siRNAs in 3 parallel measurements. Scrambled siRNA and non-transfected HeLa cells were used as controls for the experiments. The siRNA silencing revealed a remarkably significant change in the expression of 12 genes. These genes are involved in regulation of cytoskeletal function and trafficking, muscle function, and steroid biogenesis, thus, they may indirectly modify mitochondrial dynamics.
Conclusion: In DNM2 depletion samples, the downregulation of the FBLIM1, KRT13, KRT19, TMEM139, TMEM45A genes are involved in cytoskeletal function and trafficking, so they might be indirectly modify mitochondrial dynamics. TNNC1 plays a major role in the regulation of muscle function, so the decrease in expression found may be related to the formation of centronuclear nuclei. CYP4F3 gene expression was also significantly decreased. It is a monooxidase involved in cholesterol and steroid biosynthesis. Its relationship with DNM2 is currently in question. It should be noted that there are a large number of endocrine abnormalities in DNM2 positive patients, which may be explained by dysfunction of steroid biogenesis.
The study was supported by the Semmelweis University StartUp, NKFIH_ 132812 and UNKP-21-5 grants.