Study On MiR-140-5p Affecting Human Endometrial Receptivity Via Targeting VEGF

PurposeEndometrial receptivity(ER)measures the ability of endometrium to allow the location,adhesion,and invasion of blastocyst.This process was controlled by complex mechanisms.The gradually improvement of Assisted Reproductive Technology(ART)significantly increases the rate of clinical pregnancy,but the reduction of ER is still an essential factor to hinder the blastocyst implantation.ER is affected by multiple factors and vascular endothelial growth factor(VEGF)plays a crucial role in early pregnancy in many species.The changes of VEGF levels can severely affect the rate of blastocyst implantation.Therefore,the study on the regulation of ER by VEGF contributes to uncover potential biomarkers and effective treatments.Here,I examined the expression of miR-140-5p and VEGF in patient suffering repetitive failure of blastocyst implantation and generated the epidermal cell lines of human endometrium.By analyzing the expression level and cellular functions,I demonstrated the effects of progesterone/miR-140-5p/VEGF on epidermal cells of endometrium and uncovered the potential application of miR-140-5p as a molecular marker of the receptivity of endometrium.The finding of miR-140-5p also provided a potential target for improving the receptivity of endometrium.Materials and methods1.Expression of VEGF in endometrial epithelial cells(EEC)of repreated implantation failure patients and its effects on human EEC Endometrial tissues were collected from 20 patients with repeated in vitro fertilization-embryo transfer failure and 20 successful pregnancy,but suffered abnormal abortion due to embryonic chromosome.Firstly,Western blotting and qRT-PCR were used to examine the protein and mRNA levels of VEGF in patients' EECs.The effects of abnormal levels of VEGF on the function of human EECs were examined by CCK-8 assay and colony formation assay.In further,the protein levels of LIF in patients' EECs were exmanied by using Western blotting.Finally,qRT-PCR,western blotting,and flow cytometry were used to examine the effects of mis-regulation of VEGF on the alternations of cell cycle related facors such as CCNB1,CCND1,and CDK2.2.miR-140-5p affects the proliferation of human EECs via targeting VEGF Firstly,the mRNA of VEGF was predicted as the potential target of miR-140-5p.To verify the targeting ability of miR-140-5p to VEGF,we performed luciferase reporter assay.The expression levels of miR-140-5p in human endometrial tissues of 20 patients with failed in vitro fertilization-embryo transfer and 20 patients with successful in vitro fertilization-embryo transfer were detected by qRT-PCR.The effect of aberrant expression of miR-140-5p on VEGF levels were examined by qRT-PCR and Western blotting.The effects of aberrant expression of miR-140-5p on the activity of EEC were examined by CCK-8 assay and colony formation assay.3.Effect of progesterone on human EECs EEC were treated with either different concentrations(0,1,5 ? 10 nmol/L)of progesterone or 10nom/L progesterone for 12,24 and 36 h,and the activity of EEC was detected by CCK-8 assay and colony formation assay.The expression levels of miR-140-5p and VEGF were quantified by qRT-PCR after treating progesterone 10 nmol/L for 24 h.Results1.Expression of VEGF in EECs of repreated implantation failure patients and its effects on human EECsThe expression of VEGF was significantly down-regulated in endometrial tissues of patients with repeated failed in vitro fertilization-embryo transfer(P < 0.001).Overexpression or suppression of VEGF can promote or inhibit proliferation of EECs,respectively.Compared with control group,the expression of LIF protein in EEC is decreased in patients with endometrial receptivity.And,the protein levels of LIF were positive related to the levels of VEGF.Overexpression of VEGF significantly increased the mRNA and protein levels of CCNB1,CCND1,and CDK2(P < 0.01 or P <0.05),while knockdown of VEGF decreased the mRNA and protein levels of these three cell cycle-related factors(P < 0.05).2.miR-140-5p affects the proliferation of human EECs via targeting VEGFThe expression of miR-140-5p was up-regulated in endometrial tissues of patients with failed in vitro fertilization-embryo transfer.VEGF is a target gene of miR-140-5p.Overexpression of miR-140-5p inhibited the luciferase activity of wild-type VEGF 3'UTR.,but not in the mutant VEGF 3'UTR.Overexpression and suppression of miR-140-5p significantly inhibited and promoted proliferation of EECs,respectively(P < 0.05).Overexpression of miR-140-5p significantly inhibited the expression of VEGF mRNA and protein(P < 0.05),while overexpression of VEGF overcomed the inhibiting effects of miR-140-5p and promoted cell viability(P < 0.01).3.Effect of progesterone on human EECsProgesterone significantly promoted the activity of EEC in dose-and time-dependent manner(P < 0.05,P < 0.01 or P < 0.001).The formation of progesterone-treated EEC was significantly increased(P < 0.01).In EEC,the application of progesterone significantly suppressed the level of miR-140-5p and activated the expression of VEGF.(P < 0.05,P < 0.01).Conclusions1.VEGF is down-regulation in endometrial tissues of patients with repeated failed in vitro fertilization-embryo transfer.VEGF can promote EEC proliferation and cell cycle.The level of LIF protein expression in EEC and endometrial tissues of patients with repeated failed is affected by the level of VEGF.2.miR-140-5p is highly expressed in endometrial tissues of patients with repeated failed in vitro fertilization-embryo transfer.VEGF is the target gene of miR-140-5p,and miR-140-5p can inhibit the proliferation of EEC by targeting VEGF.3.The application of progesterone effectively suppresses miR-140-5p and promotes the expression of VEGF and the proliferation of EEC.