Effects Of MFF On Mitochondrial Function And Cell Proliferation-Aopotosis Of Hypoxia-induced Pulmonary Artery Smooth Muscle Cells And Its Molecular Mechanism

ObjectiveMitochondrial dynamics and quality control play a central role in the maintenance of the proliferation-apoptosis balance,which is closely related to the progression of pulmonary arterial hypertension(PAH).However,the exact mechanism of this balance remains unknown.In the present study,we constructed a PAH model in vitro by hypoxia stimulation PASMCs,and the role of mtochondrial fission factor(MFF)in the progression of PAH and its molecular mechanism were also discussed.This study aims to provide a theoretical basis for the prevention and treatment of PAH.MethodsTo establish a PAH model vitro,PASMCs were starved at DMEM medium without serum for 24h,and then cells were incubated in hypoxic condition of 92%N2,5%CO2 and 3%O2 for 48h.The expression of genes and proteins were determined by qRT-PCR and western bolt assays.Cell proliferation-apoptosis balance was tested by MTT,EdU and TUNEL assays.JC-lwas used to analyze the mitochondrial membrane potential.The mitochondrial perimeter was evaluated using MitoTracker Red staining.The ROS production was determined by using fluorescent dye mito-SOX.The ATP level and caspase 3 activities were determined using ATP assay kit and caspase activity detection kit.The molecular interaction was validated by dual luciferase reporter assay.Results1.Compared to control group,cell viability of PASMCs exposed in hypoxia were increased,meanwhile apoptosis were decreased.Mitochondrial ROS level and mitochondrial fission were increased,while mitochondrial membrane potential,ATP generation and mitochondrial perimeter were decreased(P<0.05).2.Both mRNA and protein levels of MFF were increased in hypoxia-treated PASMCs compared to that in control group.MFF was silenced in hypoxia-treated PASMCs by transfection of si-MFFs.As for hypoxia-treated PASMCs,MFF silencing reduced the excessive cell viability and proliferation,ROS release,and mitochondrial fission but increased apoptosis,mitochondrial membrane potential,content of ATP,and mitochondrial perimeter(P<0.05).3.SIRT1 and SIRT3 were decreased in hypoxia-treatment PASMCs,MFF silencing promoted the expression and the effect was eliminated by knockdown of SIRT1/3.Proliferative assays showed that silencing of SIRT1/3 reversed the inhibitory effects on cell viability and proliferation mediated by MFF knockdown.Compared to Hyp+si-MFF group,the protective effects of MFF silence on mitochondria functions were eliminated by SIRT1/3 co-silencing,presenting as the increase of ROS release and the decrease of mitochondrial membrane potential and ATP generation(P<0.05).SIRT1/3 inhibition eliminated the inhibitory effects of MFF knockdown on mitochondrial fission and the increase of mitochondrial perimeter,SIRT1/3 inhibition reversed the promotion effects of si-MFF on cell apoptosis and caspase 3 activity(P<0.05).4.Starbase online database(http://starbase.sysu.edu.cn/)predicted the binding site between miR-340-5p and MFF 3’-UTR.After bioinformatics prediction,the low expression of miR-340-5p in hypoxia-induced PASMCs was detected.MiR-340-5p expression was enhanced after transfection with miR-340-5p mimics as showed by qRT-PCR.assay.Dual luciferase reporter gene assay displayed that luciferase activity in MFF-wt group was suppressed following treated with miR-340-5p mimics,while the luciferase activity in the MFF-mut group remained no significant difference(P<0.05).Both mRNA and protein level of MFF were downregulated after miR-340-5p overexpression,while the protein levels of SIRT1 and SIRT3 were elevated.5.QRT-PCR results displayed that miR-340-5p mimics transfection increased miR-340-5p expression in hypoxia-induced PASMCs,and overexpression of MFF abolished the inhibitory effect of miR-340-5p on MFF expression(P<0.05).MiR-340-5p overexpression decreased the excessive viability and proliferation of hypoxia-treated PASMCs,while the effects were significantly reversed by co-overexpression of MFF.MiR-340-5p overexpression reduced ROS release,mitochondrial fission,and elevated mitochondrial membrane potential,mitochondrial perimeter,content of ATP,TUNEL-positive cell rate and caspase 3 activity of hypoxia-treated PASMCs by downregulating of MFF level,while MFF overexpression obviously reduced these effects.Conclusion1.The mitochondrial dysfunctions and proliferation-apoptosis imbalance of hypoxia-treated PAMSCs were mediated by MFF depended on inhibition of SIRT1/3 signaling.2.miR-340-5p regulated MFF-SIRT1/3 axis to improve mitochondrial homeostasis and proliferation-apoptosis imbalance of hypoxia-treated PASMCs...