Mechanism Of DGUOK Regulating Mitochondrial Function

DGUOK is a rate-limiting enzyme in the sputum deoxynucleotide rescue synthesis pathway and play an important role in maintaining the stability of the mitochondrial deoxyribonucleoside triphosphate pool.Mutations in the DGUOK gene cause mitochondrial DNA depletion,which can cause respiratory chain dysfunction and lead to a variety of diseases.This experiment hopes to construct mitochondria from the two experimental models of cells and mice by DGUOK knockdown,using q PCR,Blue native polyacrylamide gel electrophoresis(BN-PAGE),Western Blot,Immunofluorescence Staining,and Oxygen Consumption Rate determination.From the morphology to the functional level,we explored what functions DGUOK regulates mitochondria and understand its mechanism of action.Through experimental research,the following results were obtained:1)DGUOK affects the number and location of mitochondria in non-small cell lung cancerThe mitochondrial DNA copy number of DGUOK knock-out cell was decreased by q PCR,the m RNA levels of MT-ND1 and MT-ATP6 genes constituting mitochondria were not significantly different,and the m RNA level of MT-ND2 was significantly up-regulated.The number of mitochondria was significantly decreased under the laser scanning confocal microscope,and it was mostly gathered around the nucleus.2)DGUOK affects the function of complex I in H1650 cancer cellsThe DGUOK knock-out cells were detected by BN-PAGE,and the level of complex I on the mitochondrial respiratory chain was decreased.The protein expression of NDUFB8was decreased by Western Blot,but the m RNA level was unchanged.It is inferred that DGUOK affects the function of complex I on the mitochondrial respiratory chain.3)DGUOK affects NMNAT2 expression and mitochondrial function in cancer cellsThe effect of DGUOK on mitochondrial oxidative phosphorylation was indirectly explored by using Agilent Seahorse XF to detect mitochondrial oxygen consumption rate(OCR).Subsequently,q PCR and Western Blot were used to detect the core enzyme in NAD~+biosynthesis,nicotinamide mononucleotide adenylyl transferase(NMNAT),and It was found that the m RNA and protein expression levels of NMNAT2 in DGUOK knock-out H1650 cell were significantly down-regulated.4)DGUOK regulates NMNAT2 independent of mitochondrial complex I functionFirst,the m RNA expression level of NMNAT2 in the functionally blocked cells of the complex I after rotenone treatment was detected by q PCR.Compared with the untreated positive control cells,the NMNAT2 m RNA level was not significantly down-regulated as in the DGUOK knockout cells.5)Effect of DGUOK on mitochondrial complex I in mouse liver tissueA mouse model of Dguok knock-out was bred,and the mt DNA copy number in the liver tissue of the Dguok knock-out mouse was found to be decreased by q PCR.In contrast to the cancer cell system,Dguok deletion reduces the m RNA levels of the mitochondrial genes mt-Nd1 and mt-Co1 that make up the respiratory chain.It was found by Western Blot that the expression level of NDUFB8 and NDUFA10 encoded by the nuclear gene of the composite complex I was decreased.It is indicated that Dguok deletion in mouse liver tissue also affects mitochondrial complex I.This study shows that DGUOK affects the number and location of mitochondria in non-small cell lung cancer,and affects the function of mitochondrial respiratory chain complex I.It is found that DGUOK affects the oxygen consumption rate of cells and regulates the m RNA and protein expression of NMNAT2.DGUOK play a role in mitochondrial metabolism,but its regulation of NMNAT2 is independent of the function of complex I.DGUOK deletion results in mitochondrial DNA depletion,and the protein expression of mitochondrial respiratory chain complex I in Dguok knockout mouse liver model reduce.In summary,this paper explored the mechanism of DGUOK regulation of mitochondrial function and provided data support and theoretical reference for understanding the cerebral hepatic mitochondrial exhaustion syndrome associated with DGUOK.