孕早期增塑剂DEHP暴露降低长链非编码RNA RP24-315D19.10表达影响子宫内膜蜕膜反应

Plasticizer DEHP exposure in early pregnancy affects the endometrial decidualization in mice through reducing lncRNA RP24-315D19.10 expression

目的:探索孕早期增塑剂邻苯二甲酸二(2-乙基)己酯(DEHP)暴露对小鼠子宫内膜蜕膜反应的影响及长链非编码RNA RP24-315D19.10的作用。方法:构建DEHP(1000 mg·kg^(-1)·d^(-1))暴露的早孕小鼠模型,取妊娠第6天子宫,通过苏木精-伊红染色和免疫荧光检测其对小鼠子宫内膜蜕膜区病理变化及相关分子表达。构建DEHP暴露(0.1、0.5、2.5、12.5、62.5μmol/L)的原代小鼠子宫内膜基质细胞体外蜕膜诱导模型,通过光学显微镜和鬼笔环肽染色观察细胞形态变化,免疫荧光、实时逆转录PCR、蛋白质印迹法检测蜕膜反应标志蛋白的表达情况。实时逆转录PCR分别检测RP24-315D19.10在蜕膜组织和蜕膜细胞中的表达。利用在线工具lnc Locator和RNA荧光原位杂交检测RP24-315D19.10的细胞定位,并运用在线工具Anno Lnc2预测与RP24-315D19.10相结合的微RNA(mi RNA)。结果:DEHP暴露组的胚胎着床点数、子宫湿重、子宫面积显著低于空白对照组,蜕膜反应标志蛋白基质金属蛋白酶、同源异型框A10的表达也明显少于空白对照组(均P<0.05)。随着DEHP浓度增加,蜕膜细胞中dtprp的表达逐渐降低。2.5μmol/L DEHP暴露组中基质细胞向蜕膜细胞的形态转化受到明显抑制,蜕膜反应标志蛋白同源异型框A10、骨形成蛋白2及增殖细胞核抗原表达水平显著低于空白对照组(均P<0.05)。DEHP暴露的蜕膜组织和蜕膜细胞中RP24-315D19.10的表达显著减少(均P<0.05)。RP24-315D19.10主要定位于细胞质,与45个mi RNA有潜在结合位点,其中mi R-138-5p、mi R-155-5p、mi R-183-5p及mi R-223-3p与子宫内膜蜕膜反应有关。结论:孕早期DEHP暴露导致小鼠蜕膜反应损伤可能与RP24-315D19.10表达下调有关。

Objective:To explore the effect of exposure to di(2-ethyl)hexyl phthalate(DEHP)in early pregnancy on endometrial decidualization in mice and its relation with lncRNA RP24-315D19.10-.Methods:Early pregnancy mice were exposed to DEHP(1000 mg·kg·1d-1)to construct the model.The uterus was collected on day 6 of pregnancy to detect its effect on decidualization by HE staining and immunofluorescence.A decidualization induction model of mouse endometrial stromal cells exposed to DEHP(0.1,0.5,2.5,12.5,62.5μmol/L)was constructed.The changes of cell morphology were observed by light microscopy and phalloidin staining,and the expression of decidual reaction related molecular markers were detected by immunofluorescence,realtime RT-PCR and Western blotting.The expression of RP24-315D19.10 in decidua tissue and cells was detected by realtime RT-PCR.Cellular localization of RP24-315D19.10 was determined by lncLocator database and RNA FISH.AnnoLnc2 database was used to predict miRNAs bound to RP24-315D19.10.Results:The number of embryo implantation sites,uterine weight and uterine area were significantly lower in the DEHP exposed group than those in the control group,and the expression of the decidual reaction related molecular markers matrix metalloprotein 9 and homeobox A10 in the DEHP exposure group were also significantly lower than those in the control group(all P<0.05).With the increase of DEHP concentration,the expression of dtprp in decidua cells was gradually decreased.2.5μmol/L DEHP exposed stromal cells failed to be fully decidualized in vitro,and phalloidin staining showed abnormal cytoskeleton morphology.The expression levels of homeobox A10,bone morphogenetic protein 2 and proliferating cell nuclear antigen in the DEHP exposure group were significantly lower than those in the control group(all P<0.05).The expression of RP24-315D19.10 in DEHP exposed decidua tissue and cells was significantly reduced(both P<0.05).RP24-315D19.10 is mainly localized in the cytoplasm and RP24-315D19.10 might bind to 45 miRNAs,among them,miR-138-5p,miR-155-5p,miR-183-5p and miR-223-3p were associated with endometrial decidualization.Conclusion:DEHP exposure in early pregnancy may impair endometrial decidualization,and the damage may be associated with the downregulation of RP24-315D19.10.