The Study On The Effect And Mechanism Of MiR-484 In Regulating Ovarian Oxidative Stress Injury

Objective: To explore the role and mechanism of miR-484 in oxidative stress injury in ovaries and granulosa cells via mouse and granulosa cell oxidative stress models.Methods:(1)The mouse and granulosa cell line SVOG were treated with 3NP to induce oxidative stress.The expression levels of miR-484 were measured in ovaries and granulosa cells under oxidative stress conditions.(2)(1)The miR-484 mimic/inhibitor were transfected into granulosa cells to overexpress or inhibit miR-484,and the biological function of granulosa cells were assessed,including cell viability,cell proliferation,the contents of ROS,mtROS,MDA,SOD and GSH-Px,mitochondrial function and cell apoptosis.(2)Ovarian function was evaluated after intra-ovarian injection of AAV-mediated miR-484 inhibition under oxidative stress conditions.The body weight,ovarian weight and estrous cycle of mice were recorded,ovarian morphology was assessed,the levels of E2,FSH and AMH in serum were measured,the levels of ovarian ROS and MDA,SOD and GSH-Px were measured,apoptosis and proliferation of ovarian granulosa cells were evaluated.(3)(1)Bioinformatics analyses were performed to predict the upstream lnc RNA(LINC00958)of miR-484.After the granulosa cells were transfected with miR-484 mimic,RNA sequencing was performed to find the target gene(SESN2)of miR-484.(2)The interaction of miR-484 to LINC0098 and SESN2 was verified using dual luciferase reporter assays and granulosa cell function assay.(4)Granulosa cells transfected with miR-484mimic/inhibitor were co-cultured with GV oocytes.The numbers of oocytes at different developmental stages and abnormal oocytes were counted,and the levels of ROS,mtROS,mitochondrial function,spindle morphology,early apoptosis and chromosome aneuploidy of co-cultured oocytes were measured.Results:(1)The expression levels of miR-484 were significantly increased in 3NP-induced oxidative stress model of ovary and granulosa cells.(2)(1)Overexpression of miR-484 in granulosa cells weakened cell viability and cell proliferation,increased cell ROS,mtROS and MDA levels,decreased SOD and GSH-Px activities,exacerbated abnormal mitochondrial morphology,reduced mitochondrial membrane potential and ATP level,and raised apoptosis level.(2)Inhibiting the expression of endogenous miR-484 reversed the oxidative stress damage of granulosa cells,and inhibiting the expression of ovarian miR-484 improved the oxidative stress-induced ovarian dysfunction,including the estrous cycle,follicle development and hormone levels,reduced the apoptosis index and increased the proliferation ability of ovarian granulosa cells.(3)LINC00958/miR-484/SESN2 pathway mediated oxidative stress of granulosa cells by regulating cell viability,cell proliferation,mitochondrial function and apoptosis.(4)Oocytes co-cultured with miR-484 overexpressed-granulosa cells showed reduced maturation rate,abnormal proportion,increased levels of ROS and mtROS in oocytes,impaired mitochondrial function,increased rate of early apoptosis,abnormal spindle and chromosomal aneuploidy.Conclusions: miR-484 regulated ovarian oxidative stress by mediating granulosa cell biological function,the potential mechanisms could the downregulation of LINC00958 and its direct binding with SESN2 under oxidative stress conditions in granulosa cells,which in turn damaged mitochondrial function and compromised the mitochondrial-related apoptosis signaling pathway.Inhibition of miR-484 expression enhanced the antioxidant effect of ovaries and granulosa cells.