MiR-133b Reverses The Hydrosalpinx-induced Impairment Of Endometrial Receptivity And Embryo Implantation By Down-regulating SGK1

Object:The aim of this study was to assess the role of miR-133b in the hydrosalpinx-induced impairment of endometrial receptivity and embryo implantation.Methods:The expression of SGK1 and HOXA10 protein and miR-133b in the mid secretory endometrium of patients with hydrosalpinx were analyzed by quantitative real-time PCR or Western blot analysis.Western blotting and quantitative real-time PCR were also used to detect the expression of miR-133b,SGK1 and HOXA10 in Ishikawa cells treated with hydrosalpinx for 24h or 48h.Luciferase assay,real-time PCR and Western blot analysis was used to demonstrate that miR-133b directly targeted SGK1 through translational repression and SGK1 mediated HOXA10 expression by CREB ser133 phosphorylation.The effect of miR-133b,SGK1,HOXA10 and hydrosalpinx on embryo implantation was evaluated using a BeWo spheroid attachment assay.Furthermore,the BeWo spheroid attachment assay was performed to analyze the role of miR-133b and HOXA10 in rescuing the embryo implantation impairment induced by hydrosalpinx.Results:miR-133b and HOXA10 was down-regulated whereas SGK1 was up-regulated in the mid secretory endometrium of patients with hydrosalpinx.Ishikawa cells treated with hydrosalpinx for 24h had a 1.9-fold higher expression of SGK1,while treated for 48h had a 1.8-fold higher expression of SGK1 and an approximately 60%decrease in HOXA10 expression.Furthermore,miR-133b bound to the 35 untranslated region(39 UTR)(GGACCAA)of SGK1 mRNA and significantly reduced SGK1 expression and increased HOXAIO expression in Ishikawa cells;when endogenous miR-133b expression decreased by inhibitors,increased SGK1 by 5-fold and repressed HOXA10 to 50%.Simultaneously,miR-133b overexpression in Ishikawa cells significantly reduced CREB expression and CREB phosphorylation(ser133);whereas SGK1 overexpression increased CREB expression and CREB phosphorylation(ser133)and reduced HOXA10 expression.Knocking down CREB in Ishikawa cells showed that SGK1 regulated CREB-mediated HOXA10 protein expression.Embryo adhesiveness was promoted by miR-133b and HOXA10,whereas inhibited by SGK1.Furthermore,over-expression of miR-133b and HOXA10 could reverse the impairment of embryo adhesiveness mediated by hydrosalpinx.Conclusion:Our present findings suggest that miR-133b improves endometrial receptivity and embryo implantation by reducing SGK1 expression,leading to reverses the hydrosalpinx-induced impairment of endometrial receptivity and embryo implantation.