| Objective:An earlier study by our group found that miR-221-3p expression was significantly reduced in ovarian granulosa cells（GCs）of older women with diminished ovarian reserve（DOR）compared with younger women with normal ovarian reserve（NOR）.This study aimed to explore the effect of miR-221-3p on GCs apoptosis and to reveal the possible mechanisms of miR-221-3p involvement in the pathogenesis of DOR.To explore the efficacy and possible microscopic mechanisms of electroacupuncture in the treatment of DOR.Methods:Part I:The target gene of miR-221-3p was firstly identified by bioinformatics prediction and dual luciferase reporter assay.Then the expression of miR-221-3p was altered by transfection of KGN cells with miR-221-3p mimics,miR-221-3p inhibitor and miRNA NC,respectively.The expression of miR-221-3p and its target gene in each group was detected by quantitative real-time polymerase chain reaction（q RT-PCR）and Western blot,and the effect of miR-221-3p on apoptosis of GCs was detected by flow cytometry.Finally,the expression of target gene in GCs of DOR and NOR patients were detected.Part II:66 DOR patients with kidney deficiency were randomly divided into the experimental group and the control group,with 33 cases in each group.The experimental group was treated with electroacupuncture intervention for 2 cycles until the day of triggering,while the control group was treated with the same controlled hyperovulation protocol as the experimental group only.Selection of points:Guanyuan,Sanyinjiao（bilateral）,Zigong（bilateral）,Zhongji.Before and after treatment,we counted the changes in the TCM syndrome score.The days of gonadotropin（Gn）use,Gn dosage,the level of estradiol（E2）and progesterone（P4）on the day of ovulation triggering,the number of oocytes obtained,the number of 2PN,the number of embryos,the number of high-quality embryos and the rate of high-quality embryos were analyzed.The expression level of miR-221-3p and its target gene in GCs of the two groups was determined by q RT-PCR and Western blot.Results:Part I:1.Forkhead box transcription factor O1（FOXO1）was the target gene of miR-221-3p,and BCL2L11 was not the target gene of miR-221-3p;2.Compared with the miRNA NC group,the apoptosis rate was significantly lower in the miR-221-3p mimics group and increased in the miR-221-3p inhibitor group,both with statistically significant differences（P<0.05）;3.The level of miR-221-3p was negatively correlated with the mRNA and protein levels of FOXO1;4.The mRNA and protein levels of FOXO1 in the DOR group were significantly higher than those in the NOR group（P<0.05）.Part II:1.The E2level on the day of ovulation triggering was significantly higher in the experimental group than that in the control group（P<0.05）;2.The TCM syndrome score of kidney deficiency was significantly decreased in the experimental group after treatment（P<0.05）.There was no statistically significant difference in the TCM syndrome score of kidney deficiency in the control group before and after treatment（P>0.05）;3.The number of 2PN,the number of embryos,the number of high-quality embryos and the rate of high-quality embryos were significantly higher in the experimental group compared with those in the control group（P<0.05）,but no significant difference was observed in the number of oocytes obtained in the two groups（P>0.05）;4.Compared with the control group,miR-221-3p expression was significantly up-regulated and FOXO1mRNA and protein expression were significantly down-regulated in the GCs of the experimental group,and the differences were statistically significant（P<0.05）.Conclusions:1.In DOR patients,low expression of miR-221-3p may be involved in DOR development by upregulating FOXO1 expression to cause excessive apoptosis of GCs.2.Electroacupuncture significantly improved clinical symptoms,increased the number of 2PN,the number of embryos,the number of high-quality embryos and the rate of high-quality embryos in DOR patients with kidney deficiency.Besides,electroacupuncture resulted in upregulation of miR-221-3p expression and downregulation of FOXO1 expression.3.The improvement of oocyte and embryo quality in DOR patients by electroacupuncture may be related to the suppression of FOXO1 expression by upregulation of miR-221-3p expression,thereby attenuating the over-apoptotic GCs. |
