Objective To explore the effect of miR-543 in mediating the biological functions of ectopic endometrial stromal cells(EESCs)by targeting TWIST1.Methods The reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used to determine the expression level of miR-543.The cell counting kit-8(CCK-8)assay was utilized to evaluate the proliferation ability of EESCs.Transwell assay was conducted to assess the migration and invasion capabilities of EESCs.Flow cytometry was used to detect the apoptotic rate of EESCs.Additionally,the targeting relationship between miR-543 and TWIST1 was confirmed through a dual-luciferase reporter assay.Results Compared with the normal group,the expression of miR-543 in endometrial tissues of the EM group was decreased,while the expression of TWIST1 mRNA was increased(P<0.05).Compared with the NC mimics group,the transfection rate of miR-543 mimics group was increased(P<0.05).Compared with the NC mimics group,the miR-543 mimics group showed a lower OD450 value(P<0.05).Compared with the NC mimics group,the number of migrations and invasions in the miR-543 mimics group was decreased(P<0.05).Flow cytometry analysis indicated that the apoptosis rate in the miR-543 mimics group was increased compared with the NC mimics group(P<0.05).Compared with the NC mimics group,the luciferase activity of TWIST1wt in the miR-543 mimics group was decreased(P<0.05).Compared with the NC mimics group,the expression of TWIST1 protein in the miR-543 mimics group was decreased(P<0.05).Compared with the NC mimics group,the miR-543 mimics+Vector group showed a lower OD450 value(P<0.05).Compared with the miR-543 mimics+Vector group,the miR-543 mimics+pcDNA-TWIST1 group showed a higher OD450 value(P<0.05).Compared with the NC mimics group,the number of migrations and invasions in the miR-543 mimics+Vector group was decreased(P<0.05),and the apoptosis rate was increased(P<0.05).Compared with the miR-543 mimics+Vector group,the number of migrations and invasions in the miR-543 mimics+pcDNA-TWIST1 group was increased(P<0.05),and the apoptosis rate was decreased(P<0.05).Conclusion Overexpression of miR-543 inhibits the proliferation,migration,and invasion abilities of EESCs by regulating TWIST1,suggesting that miR-543 may be a potential therapeutic target for endometriosis.