MiR-335 Promotes Ferroptosis By Targeting Ferritin Heavy Chain 1 In In Vivo And In Vitro Models Of Parkinson’s Disease

ObjectiveIn this study,we established in vivo and in vitro models of PD induced by 6-OHDA to explore the regulatory effect of miR-335,which targets FTH1 in PD ferroptosis.Methods1.In vivo experimentsThe PD rat model was established by injecting 6-OHDA into dual targets(ventral tegmental area and substantia nigra pars compacta).Male Sprague-Dawley rats were randomly assigned to 4 groups:Control(saline),Model(6-OHDA),Mimic(6-OHDA+miR-335 mimic),or Inhibitor(6-OHDA+miR-335 inhibitor).TH is a protein marker of PD.GPX4 is a vital marker protein of ferroptosis.FTH1 is a target protein of PD ferroptosis.Brain tissue was harvested,and immunohistochemistry was performed to detect the expression of TH,GPX4 and FTH1.Western Blot(WB)was used to detect the protein expression of TH,GPX4 and FTH1 in each group.RT-qPCR was used to validate the differential expression of miRNAs that may regulate FTH1 in the SN of control and model rats.2.In vitro experimentsDual luciferase reporter assay was used to determine the interaction between miR-335 and 3’UTR of FTH1 mRNA.PC12 cells were induced by 6-OHDA to establish a PD cell model.To verify that miR-335 promotes ferroptosis by targeting FTH1 in PD,PD cells were transfected with miR-335 mimic,miR-335 inhibitor,miR-335 mimic NC,miR-335 inhibitor NC,siRNA-FTH1 and siRNA NC and detect the expression of TH,FTH1 and GPX4.Cellular iron concentration assay,intracellular ferrous ion imaging,lipid peroxidation generation detection,cellular ROS detection,mitochondrial membrane potential test were also performed.ResultsIn vivo and in vitro results showed that the expression of ferroptosis marker protein GPX4 and PD marker protein TH were decreased in the Model group,which was associated with reduced ferritin heavy chain 1(FTH1)expression and upregulation of miR-335.In both the in vivo and in vitro models,miR-335 mimic led to lower FTH1 expression,exacerbated ferroptosis,and enhanced PD pathology.Luciferase 3’untranslated region reporter results identified FTH1 as the direct target of miR-335.FTH1 silencing in 6-OHDA induced cells strengthened the effect of miR-335 on ferroptosis and enhanced PD pathology.Mechanistically,miR-335 enhanced ferroptosis through degradation of FTH1 to increase iron release,lipid peroxidation and ROS accumulation and downregulate mitochondrial membrane potential(MMP).ConclusionOur findings reveal that miR-335 promotes ferroptosis by targeting FTH1 in in vitro and in vivo models of PD,providing a potential therapeutic target for the treatment of PD...