The Mechanism Of FUNDC1 On The Proliferation Of Pulmonary Endothelial Cells By Rwgulating The Level Of Mitochondrial Division In Mice

Objective To explore the mechanism of FUNDC1 on the proliferation of pulmonary artery endothelial cells by regulating the level of mitochondrial division.Methods Mouse pulmonary artery endothelial cell line was used as model for the experiment.(1)In order to clarify whether FUN14 domain containing 1(FUNDC1)can regulate the mitochondrial division level of mouse pulmonary endothelial cells(PAECs)and affect their proliferation,PAECs was divided into normoxia control group,tumor necrosis factor(TNF)-α group,TNF-α+no-load group,TNF-α+ overexpression FUNDC1 group,TNF-α+ no-load +Mdivi-1 group,TNF-α+ overexpression FUNDC1+Mdivi-1 group.The mRNA and protein expression levels of FUNDC1,microtubule-associated protein1 light chain 3(LC3B),P62,dynamin-related protein 1(DRP1)and proliferating cell nuclear antigen(PCNA)in each group were detected by qRT-PCR and Western blot.The proliferation level of mouse PAECs was detected by 5-ethynyl-2’-deoxyuridine(EDU)kit.Mitochondrial membrane potential kit and mitochondrial reactive oxygen kit were used to detect mitochondrial membrane potential and reactive oxygen species in mouse PAECs.Autophagy of mitochondria in mouse PAECs was observed by immunofluorescence staining and confocal microscopy.Mitochondrial morphological changes in mouse PAECs were observed by transmission electron microscopy.(2)To determine whether Ubiquitin-Specific peptidase 15(USP15)is involved in the regulation of PAECs proliferation by FUNDC1,they were divided into NC group,TNF-α group,TNF-α+ adenovirus no-load group,TNF-α + overexpression FUNDC1 group,TNF-α + double no-load group,TNF-α +overexpression FUNDC1+siRNA no-load group,TNF-α+siUSP15+ adenovirus no-load group,TNF-α+ overexpression FUNDC1+siUSP15 group.The mRNA and protein expression levels of FUNDC1,DRP1,USP15 and PCNA in each group were detected by qRT-PCR and Western blot.The proliferation level of mouse PAECs was detected by EDU kit.Immunocoprecipitation combined with Western blot was used to determine whether USP15 regulated DRP1 ubiquitination.Results1.After 24 h stimulation of PAECs by TNF-α,compared with control group,FUNDC1 protein expression is decreased,PCNA,DRP1,P62 protein expression are increased,LC3 B Ⅱ/Ⅰ ratio is increased(P<0.05)in stimulation group.EDU results show that marker cells are increased in stimulation group compared with control group(P<0.05).2.Overexpression of FUNDC1 inhibits the expression of DRP1,TOM20 and PCNA in PAECs(P<0.05),and the oxidative stress response of mitochondria is weakened(P<0.05).After adding Mdivi-1,the expression of PCNA protein in PAECs continues to decrease(P<0.05).After adding proteasome MG-132,the level of DRP1 protein in PAECs is increased(P<0.05),but the mRNA expression was not significantly changed(P>0.05).3.Electron microscopy shows that compared with the control group,the number of mitochondria and the proportion of spherical mitochondria increase in the stimulation group(P<0.01).After overexpression of FUNDC1,the number of mitochondria and the proportion of spherical mitochondria decrease significantly(P<0.05).4.Overexpression of FUNDC1 inhibits the expression of USP15 protein in PAECs(P<0.05),and the expression of PCNA and DRP1 protein in PAECs continue to decrease after interference of USP15 expression(P<0.05).5.USP15 can interact with DRP1 in PAECs and prevent DRP1 degradation by ubiquitinproteasome system through deubiquitination.Conclusion FUNDC1 inhibits TNF-α-induced proliferation by inhibiting mitochondrial division of mouse pulmonary endothelial cells through USP15/DRP1 axis.