Abstract:Objective To investigate the effects of c.349G>A and c. 908T>C mutations in HARS2 gene on mitochondrial and cell function so as to reveal the pathogenic mechanism of HARS2 mutation in Perrault syndrome. Methods Recombinant expression vectors containing wild-type, c.349G>A mutant and c.908T>C mutant HARS2 were constructed. The plasmid was transfected into HEK 293T cells using lentivirus packaging system. Stably transfected cell lines were screened using puromycin. Meanwhile, the cell line transfected with empty virus vector was established as the control. The mitochondrial function and cell proliferation of HEK 293T cell lines in the wild group, two mutant groups and the control group were studied. Results The recombinant plasmid was mutated at the desired site, and the EGFP protein carried by the recombinant vector was expressed stably. The cell proliferation activities of the two mutant groups were lower than that of the wild group, and the absorbance at 72 h was significantly different (P<0.05). MTCO2 protein expression in c.349G>A mutant group was lower than those in the control and wild groups (P<0.05). Compared with the control group, the mitochondrial ATP relative to total cellular ATP production was decreased in both mutation groups (P<0.05). The level of mitochondrial reactive oxygen species in the c.349G>A mutant group was increased compared with the control group (P<0.05). The mitochondrial membrane potential of the two mutation groups was lower than that of the wild group (P<0.01). Conclusion The mitochondria of HARS2 c.349G>A and c.908T>C mutant cells have certain respiratory defects and the proliferation ability of the mutant cells is reduced, which may be the cytological mechanism of Perrault syndrome induced by HARS2 mutation.
