hsa-circRNA-103716靶向miR-574-5p对反复种植失败患者子宫内膜容受性的影响

The effect of hsa-circRNA-103716 targeting miR-574-5p on endometrial receptivity in patients with repeated implantation failure

目的检测hsa-circRNA-103716及miR-574-5p在人子宫内膜中的表达,探讨两者在反复种植失败患者子宫内膜容受性中的作用。方法qRT-PCR法检测70例行冻融胚胎移植反复种植失败患者(RIF组)及70例同期胚胎移植后妊娠成功患者(对照组)种植窗期子宫内膜组织及血清中hsa-circRNA-103716及miR-574-5p表达,分析其与子宫内膜容受性病理因素间的相关性。将人原代子宫内膜上皮细胞(EECs)分为NC组、hsa-circRNA-103716阴性对照(si-control)组、hsa-circRNA-103716敲减(si-circRNA)组、miR-con组、miR-574-5p过表达(mimic)组、hsa-circRNA-103716+pcDNA组和hsa-circRNA-103716+pcDNA-miR-574-5p组,CCK-8法、EdU法和细胞划痕试验检测各组细胞增殖、迁移能力,Western blot检测子宫内膜容受性相关蛋白表达;双荧光素酶报告基因法验证两者间的靶向关系。结果hsa-circRNA-103716在RIF组种植窗期子宫内膜组织及血清中表达均高于对照组,而miR-574-5p表达明显低于对照组,差异有统计学意义(均P<0.001)。hsa-circRNA-103716表达与年龄、体质量指数、流产史、不孕年限无关(P>0.05),但与子宫内膜厚度、形态、容积显著相关(P<0.05)。与si-control组相比,si-circRNA组EECs细胞活力、EdU阳性率、细胞迁移率、白血病抑制因子(LIF)、血管内皮生长因子(VEGF)、骨桥蛋白(OPN)、同源框基因A10(HOXA10)、妊娠相关蛋白A(glycodelin-A)及孕激素受体(PRs)蛋白表达量明显升高,而肿瘤坏死因子-α(TNF-α)、雌激素受体(ERs)、白细胞介素-16(IL-16)表达量明显降低,差异均有统计学意义(均P<0.05)。共转染hsa-circRNA-103716与miR-574-5p后,EECs细胞活力、5-乙炔基-2'-脱氧尿苷(EdU)阳性率、迁移能力、白血病抑制因子(LIF)、血管内皮生长因子(VEGF)、骨桥蛋白(OPN)、同源框基因A10(HOXA10)、妊娠相关蛋白A(glycodelin-A)及孕激素受体(PRs)蛋白表达量明显升高,而TNF-α、ERs、IL-16表达量明显降低,差异均有统计学意义(均P<0.05)。miR-574-5p与WT-hsa-circRNA-103716共转染后,miR-574-5pmimic组细胞荧光素酶活性低于miR-con组,差异有统计学意义(P<0.05)。结论hsa-circRNA-103716在反复种植失败患者子宫内膜中高表达,而miR-574-5p呈低表达,hsa-circRNA-103716可靶向miR-574-5p降低反复种植失败患者子宫内膜容受性。

Objective To detect the expression of hsa-circRNA-103716 and miR-574-5p in human endometrium,and explore the role of both in the endometrial receptivity of patients with repeated implantation failures.Methods Detect the expression of hsa-circRNA-103716 and miR-574-5p in endometrial tissue and serum of 70 patients with recurrent implantation failure of frozen thawed embryo transfer(RIF group)and 70 patients with successful pregnancy after embryo transfer(control group)at planting window period by qRT-PCR,and to analyze their correlation with endometrial receptive pathological factors.Human primary endometrial epithelial cells(EECs)were divided into NC group,hsa-circRNA-103716 negative control(si-control)group,hsa-circRNA-103716 knockdown(si-circRNA)group,miR-con group,miR-574-5p overexpression(mimic)group,hsa-circRNA-103716+pcDNA group,and hsa-circRNA-103716+pcDNA-miR-574-5p group,CCK-8 method,EdU method,and cell scratch assay were used to detect the proliferation and migration ability of cells in each group.Western blotting was used to detect the expression of endometrial receptivity related protein.Verify the targeting relationship between hsa-circRNA-103716 and miR-574-5p by using the dual luciferase reporter gene method.Results The expression of hsa-circRNA-103716 in the endometrium and serum of the RIF group during the implantation window period was higher than that of the control group,while the expression of miR-574-5p was significantly lower than that of the control group,with a significant difference(all P<0.001).The expression of hsa-circRNA-103716 was not related to age,body mass index,history of miscarriage,and infertility years(P>0.05),but significantly correlated with endometrial thickness,morphology,and volume(P<0.05).Compared with si-control group,EECs cell activity,EdU positive rate,cell mobility,LIF,VEGF,OPN,HOXA10,glycodelin-A and PRs protein expression levels of si-circ RNA group were significantly increased,but the expressions of TNF-α,ERs and IL-16 were significantly decreased,and the differences were statistically significant(all P<0.05).After co-transfection with hsa-circRNA-103716 and miR-574-5p,EECs cell activity,EdU positive rate,cell mobility,LIF,VEGF,OPN,HOXA10,glycodelin-A and PRs protein expression levels were significantly increased,but the expressions of TNF-α,ERs and IL-16 were significantly decreased,and the differences were statistically significant(all P<0.05).After co-transfection of miR-574-5p with WT-hsa-cicRNA-03716 into EEC cells,the luciferase activity in the miR-574-5p mimic group was lower than that in the miR-con group.The difference was significant(P<0.05).Conclusion hsa-circRNA-103716 is highly expressed in the endometrium of patients with repeated implantation failure,while miR-574-5p is low in expression,hsa-circ-RNA-103716 can reduce endometrial receptivity in patients with recurrent implantation failure by targeting miR-574-5p.