Molecular Mechanism Of Circadian Clock Gene PER1 Promoting Ferroptosis And Inhibiting The Occurrence And Development Of Oral Squamous Cell Carcinoma

Objects:Current studies have proven that the abnormal expression of the core circadian clock gene Period 1（PER1）is closely related to the occurrence and progression of multiple cancers,but the mechanism involved is largely unknown.We previously found that PER1 expression was decreased in oral squamous cell carcinoma（OSCC）tissues and was significantly correlated with the TNM clinical stage and survival time of OSCC patients.However,the specific regulatory mechanism of PER1 in OSCC remains unclear.Decreased secretion of melatonin is an important cause of the development of various cancers including OSCC,and melatonin regulates PER1 expression in various tissues.But it is unclear whether melatonin regulates PER1 in OSCC.Recent studies have shown that ferroptosis is involved in regulating the occurrence and development of OSCC,and melatonin can also regulate the ferroptosis in non-cancer diseases.But whether melatonin is involved in regulating ferroptosis in cancer has not been reported.Therefore,this study aimed to study the molecular mechanism of the regulation of circadian clock genes in OSCC,to explore the regulatory role of melatonin and PER1 in ferroptosis in OSCC,and the specific regulatory role of melatonin on PER1.Methods:The expression of ferroptosis negative regulator glutathione peroxidase 4（GPX4）was detected in paraffin sections of cancer tissues of OSCC patients with immunohistochemistry and the relationship between its expression and clinicopathological features of OSCC patients was analyzed.The expression of ferroptosis related proteins GPX4,solute carrier family 7 member 11（SLC7A11）and transferrin receptor 1（TFRC）was then detected in OSCC cells overexpressing or silencing PER1.The levels of intracellular reduced glutathione（GSH）,malondialdehyde（MDA）,reactive oxygen species（ROS）and Fe²⁺were detected,and the changes of mitochondrial morphology were observed.In the mechanism study,the half-life of the protein was detected by the CHX tracking assay,the binding of the proteins was detected by CO-IP,and the effect of the hypoxia-inducible factor 1alpha（HIF-1α）on PER1 was detected by the Ch IP assay and the dual-luciferase reporter assay.Regulation of promoter transcriptional activity.Results:In this study,we found that PER1 was negatively correlated with the expression of the key ferroptosis-regulated proteins GPX4 and HIF-1αin OSCC tissues.PER1 promotes ferroptosis depending on HIF-1αin PER1-overexpressing OSCC cells,and the opposite results were obtained in PER1-silenced OSCC cells.Subcutaneous tumor formation experiments in nude mice.The assays in vivo proved that PER1overexpression inhibits HIF-1α,promotes ferroptosis and suppresses OSCC growth.Mechanistically,PER1 binds to HIF-1αto promote HIF-1αprotein degradation,and HIF-1αbinds to the PER1 promoter to inhibit PER1transcription.That is,the binding of PER1 and HIF-1αforms the PER1/HIF-1αnegative feedback loop.In addition,we found that melatonin promotes ferroptosis of OSCC cells in vitro by regulating the PER1/HIF-1αfeedback loop.Subcutaneous tumor formation assays in nude mice.The assays in vivo have showed that melatonin increases PER1 expression,decreases HIF-1αexpression,and promotes ferroptosis to inhibit OSCC growth.Conclusions:These findings suggest that targeting the PER1/HIF-1αloop may provide a new strategy for OSCC treatment,and the development of melatonin targeting agents may be an effective method for OSCC treatment.