MicroRNA-223调控胚胎着床的机制研究

发布时间：2018-06-05 12:57

本文选题：miR-223 + 靶基因　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分miR-223调控子宫内膜上皮细胞IGF-1R的表达目的：构建miR-223过表达慢病毒载体,通过转染RL95-2上皮细胞系明确miR-223在子宫内膜上皮细胞中的靶基因。方法：利用TargetScan5.1获得miR-223的潜在靶基因信息；采用慢病毒转染体系上调RL95-2上皮细胞系中miR-223的表达,分别用real-time PCR、Western Blot检测预测靶基因mRNA和蛋白表达水平；构建miR-223的靶标克隆载体,利用双荧光素酶报告基因载体确定靶基因。结果：(1)根据TargetScan5.1预测得到miR-223的潜在靶基因IGF-1R、FOXO1、E2F1、 IKKα;(2)过表达RL95-2上皮细胞中miR-223后IGF-1R、FOXO1、E2F1、IKKα的mRNA表达均无改变,但是IGF-1R的蛋白表达明显下降；(3)双荧光素酶报告基因证实miR-223直接调节IGF-1R的表达。结论：miR-223作用在IGF-1R的3’UTR调节子宫内膜上皮细胞IGF-1R的蛋白表达。第二部分miR-223和IGF-1R在人类子宫内膜的表达规律目的：通过检测月经周期不同时期子宫内膜miR-223和IGF-1R的表达,观察人类子宫内膜上miR-223和IGF-1R的表达趋势。方法：利用Real-time PCR检测增生中期和分泌中期子宫内膜miR-223和IGF-1R mRNA的表达水平,Western Blot和免疫组化检测增生中期和分泌中期子宫内膜IGF-1R的蛋白表达水平及细胞定位。结果：(1)Real-time PCR结果显示miR-223在分泌中期子宫内膜的表达明显低于增生中期子宫内膜,而IGF-1R的mRNA在两组子宫内膜上的表达未见明显改变；(2)Western Blot结果显示IGF-1R蛋白在分泌中期子宫内膜上的表达明显高于增生中期子宫内膜；(3)免疫组化结果显示IGF-1R主要表达在子宫内膜上皮细胞上,且分泌中期子宫内膜上的表达明显高于增生中期子宫内膜。结论：miR-223和IGF-1R在人类子宫内膜上的表达呈现负相关,进一步证实了细胞实验的结论。第三部分过表达miR-223抑制胚胎的植入目的：通过体外滋养细胞球侵入模型和在体胚胎植入模型研究miR-223对胚胎植入的影响。方法：利用Bewo滋养细胞和RL95-2上皮细胞系构建内膜-滋养细胞球相互作用的体外侵入模型,观察Bewo细胞球在单层内膜上皮细胞上的侵入情况；建立小鼠妊娠模型,使用miR-223激动剂干预小鼠子宫内膜miR-223的表达,观察miR-223对胚胎植入的影响。结果：(1)在Bewo细胞球和RL95-2上皮细胞共培养体系中,过表达miR-223组Bewo细胞球的侵入率明显低于对照组,添加IGF-1可逆转miR-223对Bewo细胞球的抑制效果；(2)小鼠宫角注射miR-223激动剂干预后小鼠着床胚胎数明显下降。结论：体内及体外实验结果均证实了miR-223抑制胚胎着床,且这一抑制效应可能是通过调节IGF-1R实现的。
[Abstract]:Part one: miR-223 regulates the expression of IGF-1R in endometrial epithelial cells Aim: to construct miR-223 overexpression lentivirus vector and to identify the target gene of miR-223 in endometrial epithelial cells by transfection of RL95-2 epithelial cell line. Methods: the potential target gene information of miR-223 was obtained by TargetScan5.1, the expression of miR-223 in RL95-2 epithelial cell line was up-regulated by lentivirus transfection system, the expression level of mRNA and protein was predicted by real-time PCR Western Blot, and the target clone vector of miR-223 was constructed. The target gene was identified by double luciferase reporter gene vector. Results according to TargetScan5.1 prediction, the miR-223 potential target gene IGF-1RnFOXO1E2F1 (IKK 伪 F2F1) overexpression in RL95-2 epithelial cells showed no change in mRNA expression after miR-223, but the expression of IGF-1R protein decreased significantly (3) the double luciferase reporter gene confirmed that miR-223 directly regulated the expression of IGF-1R. Conclusion the 3'UTR of IGF-1R regulates the expression of IGF-1R protein in endometrial epithelial cells. The second part: the expression of miR-223 and IGF-1R in human endometrium Aim: to observe the expression trend of miR-223 and IGF-1R in human endometrium by detecting the expression of miR-223 and IGF-1R in endometrium at different periods of menstrual cycle. Methods: Real-time PCR was used to detect the expression of miR-223 and IGF-1R mRNA in the endometrium of metaphase hyperplasia and middle secretory period. Western Blot and immunohistochemistry were used to detect the expression and localization of IGF-1R in the middle and middle secretory phase of endometrium. Results the results of Real-time PCR showed that the expression of miR-223 in the middle secretory endometrium was significantly lower than that in the proliferative endometrium. The expression of IGF-1R mRNA in the endometrium of the two groups did not change significantly. The results of Western Blot showed that the expression of IGF-1R protein in the middle secretory endometrium was significantly higher than that in the proliferative endometrium. On the endometrial epithelial cells, And the expression of endometrial secretory medium was significantly higher than that of proliferative endometrium. Conclusion there is a negative correlation between the expression of miR-223 and IGF-1R in human endometrium, which further confirms the conclusion of cell experiment. The third part: overexpression of miR-223 inhibits embryo implantation Aim: to study the effect of miR-223 on embryo implantation by invading trophoblast ball in vitro and in vivo embryo implantation model. Methods: Bewo trophoblastic and RL95-2 epithelial cell lines were used to establish an in vitro invasion model of intima-trophoblastic interaction, to observe the invasion of Bewo cells on monolayer endometrial epithelial cells, and to establish a mouse pregnancy model. MiR-223 agonist was used to interfere the expression of miR-223 in mouse endometrium and to observe the effect of miR-223 on embryo implantation. Results in the co-culture system of Bewo cell ball and RL95-2 epithelial cell, the invasion rate of Bewo cell ball in over-expressed miR-223 group was significantly lower than that in control group. The inhibitory effect of miR-223 on Bewo cells was reversed by adding IGF-1. The number of implanted embryos in mice was significantly decreased after the injection of miR-223 agonist into uterine horn of mice. Conclusion: the results of in vivo and in vitro experiments confirm that miR-223 inhibits embryo implantation, and this inhibitory effect may be achieved by regulating IGF-1R.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R321

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