The Effects Of MiR-185-5p On Endometrial Stromal Cell Of Endometriosis Via Regulating VEGF-A

Objective:To examine the expression level of miR-185-5p in endometriosis(EMs),its influence on migration and invasion of eutopic endometrial stromal cells(ESC)in EMs,and possible molecular mechanisms.Methods:(1)qRT-PCR was used to detect the expression levels of miR-185-5p in the eutopic endometrium and ectopic endometrium among patients with III-IV EMs as well as that in the normal endometrium.(2)Scratch test and Transwell test were used to analyze the effect of miR-185-5p on the migration and invasion of ESC.(3)Western blot were used to detect the expression of VEGF-A in eutopic ESC and normal ESC.and the expression of VEGF-A after transfection of miR-185-5p-mimics and miR-185-5p-inhibitor into eutopic ESC.(4)Dual luciferase reporter gene experiment was conducted to examine the binding site between target gene VEGF-A and miR-185-5p.(5)After constructing the VEGF-A overexpression lentiviral vector,eutopic endometrium ESC were intervened(or not intervened).miR-185-5p-mimics and miR-185-5p-mimics-NC were used again to intervene.scratch test and Transwell test were used to examine the changes in the migration and invasion capabilities of ESC.Results:(1)The qRT-PCR test indicated that the expression level of miR-185-5p in the eutopic endometrium was lower than that of the control group(P<0.001),and the expression level of the eutopic ESC was lower than that of the control group too(P<0.05).(2)The Scratch and Transwell test showed that the overexpression of miR-185-5p inhibited the migration(P<0.01)and invasion(P<0.01)of eutopic ESC;while miR-185-5p knockdown promoted eutopic ESC migration(P<0.001)and invasion(P <0.001).(3)Western blot showed that the expression of VEGF-A protein in eutopic ESC was higher than that in normal ESC(P<0.001).(4)Western blot further demonstrated that compared with miR-185-5p-mimics-NC group,VEGF-A protein expression was significantly reduced after miR-185-5p-mimics intervention(P<0.01).In contrast,compared with miR-185-5p-inhibitor-NC group,the expression of VEGF-A protein in the miR-185-5p-inhibitor transfection group was significantly increased(P<0.01).(5)Dual luciferase reporter gene experiment showed that VEGF-A had a targeted binding site with miR-185-5p.(6)The Scratch and Transwell test further demonstrated that overexpression of VEGF-A significantly increased the migration(P<0.001)and invasion(P<0.01)of ESC,while miR-185-5p-mimics significantly reduced the migration(P<0.01)and invasion(P<0.001)of ESC;overexpression of VEGF-A significantly antagonized the inhibitory effect of miR-185-5p-mimics on ESC migration(P<0.01)and invasion(P<0.001).Conclusion:(1)miR-185-5p was down-regulated in the eutopic endometrium of EMs,and is involved in mediating the migration and invasion of eutopic ESC.(2)There was a targeted binding relationship between miR-185-5p and VEGF-A.miR-185-5p may inhibit the expression of VEGF-A by inhibiting the migration and invasion of eutopic ESC.