Abstract
Purpose :
The mtDNA can be classified into haplogroups that represent the ancient origins of populations. The H mtDNA haplogroup is the most common European haplogroup (~30%). The K haplogroups represents approximately 10% of the European population. Previous studies show differences in expression levels for methylation, acetylation and cancer related genes, along with different responses to amyloid beta stressor. To evaluate the differences in ferroptosis related gene expression levels in H versus K cybrids and also ARPE-19 versus Rho0 cells.
Methods :
Rho0 Cell Generation. ARPE-19 cells were treated with 50µM ethidium bromide and passaged until mtDNA was not detected. Cybrid Formation. Peripheral blood was collected in sodium citrate tubes and DNA was isolated. Using a series of centrifugation steps, platelets were isolated, suspended in Tris buffer saline , and then fused with ARPE19 cells that were deficient in mtDNA (Rho0). All experiments used cybrid cells at Passage 5. Total RNA was isolated from ARPE19 wild type and ARPE19 Rho0 cultures (H cybrids, n = 4; K cybrids, n = 5). The cDNA generated from individual RNA was used for qRT PCR analyses.The qRT PCR was performed on individual samples on an Applied Biosystems qRT PCR detection system. Primers were used to analyze 10 genes involved in Ferroptosis. All analyses were performed in triplicate. Relative fold change measured using 2(delta delta Ct).
Results :
H versus K cybrids The K cybrid had 40% lower expression levels of ASCL3 compared to the H cybrids (P<0.01, transcription factor for conversion of monounsaturated fatty acids into fatty acyl-CoAs). ARPE-19 versus Rho0 Cells - The Rho0 cells showed significantly higher levels of SLC7A11 (p<0.001, inhibits ferroptosis), TFRC (p<0.012, uptake of iron inhibits ferroptosis) and VDAC3 (P<0.01, mitochondrial porin family) but decreased levels of ASCL1 and ACSL3 (p<0.001, monounsaturated fatty acids production inhibits ferroptosis).
Conclusions :
The absence of the mitochondria (Rho0 cells) modulated the expression of some ferroptosis-related genes. Moreover, cybrids with the K mtDNA haplogroup mitochondria expressed lower levels of ASCL3, which may result in lower MUFA production compared to H cybrids.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.