miR-200c靶向调控FUT4调节子宫接受态的建立

发布时间：2018-04-28 11:36

本文选题：miR-200c + FUT4　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景与目的:1.蛋白质岩藻糖基化在子宫内膜对胚胎的接受能力方面发挥重要作用。2.岩藻糖基转移酶Ⅳ(FUT4)在哺乳动物子宫内膜上皮细胞中呈阶段特异性表达,因此被作为评价子宫内膜接受态的一个重要指标。3.micro RNAs是一种内源性非编码小分子RNA,miR-200c是miR-200家族的成员之一,在人类生殖过程中发挥作用。但是,miR-200c与FUT4的关系以及其在子宫内膜接受态的建立过程中的作用未见报道。4.本研究旨在探讨miR-200c对子宫内膜接受态的建立、胚胎着床的影响及其作用机制,阐述miR-200c对哺乳动物胚胎着床过程的调控作用。方法:1.通过Real-time PCR,Western blot,ELISA检测正常未孕、不孕症、正常早孕、流产患者血清中miR-200c、FUT4的表达。2.CCK8、平板克隆形成实验和细胞体外黏附实验检测miR-200c对细胞增殖及接受胚胎能力的影响。3.双荧光素酶基因报告实验验证FUT4是否是miR-200c的靶基因。4.通过Wnt/β-catenin信号通路进一步研究miR-200c对胚胎着床的调控作用。结果:1.PCR、ELISA和Western blot方法检测结果显示:不孕症患者血清中miR-200c的表达比正常未孕女性高,流产患者血清中miR-200c的含量比正常早孕女性血清中高,具有显著性差异(*p0.05;***p0.001)。不孕症患者血清中FUT4的表达比正常未孕女性低,流产患者血清中FUT4的含量比正常早孕女性血清中低,具有显著型差异(**p0.01;***p0.001)2.miR-200c抑制细胞增殖和胚胎黏附功能。CCK-8实验连续5天记录细胞的生长情况。转染miR-200c mimics细胞组的生长速度显著慢于对照组细胞,而转染Anti-miR-200c后,细胞的增殖能力显著高于对照组。平板克隆形成实验结果显示,miR-200c mimics转染组细胞的克隆形成数低于对照组,而转染Anti-m R-200c细胞组细胞的克隆形成数显著高于对照组,差异存在统计学意义(**p0.01;***p0.001)。细胞体外黏附实验结果显示,转染miR-200c mimics细胞组的黏附能力显著低于对照组,转染Anti-miR-200c细胞组的黏附能力显著高于对照组,差异存在统计学意义(*p0.05)。3.FUT4是miR-200c的靶基因。通过软件预测发现miR-200c与FUT4存在结合位点,因此我们构建了相应荧光素酶载体,同时构建了相应的突变型载体,与miR-200c mimics共同转染进入RL-95细胞中。转染miR-200c mimics与荧光素酶载体的RL95-2细胞中,FUT4的荧光素酶活性低于对照组(*p0.05);转染miR-200c mimics与荧光素酶载体的RL95-2细胞中FUT4的荧光素酶活性与转染突变型载体组FUT4的荧光素酶活性不存在统计学差异。Western blot结果显示miR-200c mimics转染后可显著下调FUT4的表达,当与FUT4 c DNA共转染后,又可以回调FUT4的表达。Anti-miR-200c转染后可显著上调FUT4的表达,当与FUT4 si RNA共转染后可回调FUT4的表达。细胞免疫荧光染色得到了相同的结果,miR-200c可下调FUT4的表达。4.miR-200c通过抑制Wnt/β-catenin信号通路的激活影响子宫内膜细胞的功能。转染miR-200c可抑制p-GSK3β、β-catenin的表达,其结果与使用Wnt/β-catenin通路抑制剂DKK的效果一致,当与FUT4 c DNA共转染后可重新激活p-GSK3β、β-catenin的表达。当Anti-miR-200c抑制miR-200c的表达后可激活p-GSK3β、β-catenin的表达,与FUT4 si RNA共转染后可重新抑制p-GSK3β、β-catenin的表达。结论:1.miR-200c可由血清检测,其表达量与FUT4呈负相关。2.抑制miR-200c表达可改善人内膜细胞RL95-2和胚胎细胞JAR之间的黏附。3.miR-200c对FUT4具有靶向调控作用。4.miR-200c通过靶向FUT4,抑制Wnt/β-catenin信号通路的激活,抑制子宫内膜接受态的建立。
[Abstract]:Background and purpose: 1. protein fucoylation plays an important role in the acceptance of the endometrium in the endometrium..2. fucoidase IV (FUT4) is a stage specific expression in the epithelial cells of mammalian endometrium, so it is an important indicator of the acceptance of endometrium,.3.micro RNAs is a kind of endogenous The non coding small molecule RNA, miR-200c, is one of the members of the miR-200 family and plays a role in the human reproduction process. However, the relationship between miR-200c and FUT4 and its role in the establishment of endometrial receptivity has not been reported. The purpose of.4. study is to explore the establishment of miR-200c in endometrium acceptance, the effect of embryo implantation and its work. The mechanism was used to explain the regulation of miR-200c on the implantation process of mammalian embryos. Methods: 1. Real-time PCR, Western blot, ELISA were used to detect normal unpregnancy, infertility, normal early pregnancy, the expression of miR-200c, FUT4 in the serum of abortion patients, the experiment of flat clones and the adhesion test of cells in vitro to detect the proliferation and connection of the cells by miR-200c. The effect of.3. double luciferase gene report test on the effect of embryo ability on whether FUT4 is the target gene of miR-200c,.4. can further study the regulation of miR-200c on embryo implantation through the Wnt/ beta -catenin signaling pathway. Results: 1.PCR, ELISA and Western blot method detection results show that the expression of miR-200c in the serum of the patients with infertility is more than normal The content of miR-200c in the serum of abortion patients is higher than that of normal pregnant women (*p0.05; ***p0.001). The expression of FUT4 in serum of infertility patients is lower than that of normal unpregnant women. The content of FUT4 in the serum of abortion patients is lower than that of normal early pregnant women, and there is a significant difference (**p0.01; ***p0.001) 2.mi. The growth of R-200c cells was recorded for 5 days for 5 days. The growth rate of the transfected miR-200c mimics cell group was significantly slower than that of the control group. After transfection of Anti-miR-200c, the proliferation ability of the cells was significantly higher than that of the control group. The results of the cell clone formation experiment showed that the miR-200c mimics transfection group was found. The number of cell clones was lower than that of the control group, and the number of cloned cells in the transfected Anti-m R-200c cell group was significantly higher than that of the control group, the difference was statistically significant (**p0.01; ***p0.001). The adhesion energy of the cells in vitro in vitro showed that the adhesion energy of the transfected miR-200c mimics cell group was significantly lower than that of the control group, and the transfection of the Anti-miR-200c cell group was significantly lower than that of the control group. The adhesion ability was significantly higher than that of the control group. The difference was statistically significant (*p0.05).3.FUT4 was the target gene of miR-200c. The binding site of miR-200c and FUT4 was found by software prediction. Therefore, we constructed the corresponding luciferase carrier, and constructed the corresponding mutant vector and transfected into RL-95 cells with miR-200c mimics. In RL95-2 cells infected with miR-200c mimics and luciferase carrier, the luciferase activity of FUT4 was lower than that of the control group (*p0.05), and the luciferase activity of FUT4 in RL95-2 cells transfected with miR-200c mimics and luciferase carrier was not statistically different from the luciferase activity of the mutant carrier group FUT4, and the.Western blot results showed that the activity of the luciferase activity of the FUT4 transfected with the mutant carrier group was not statistically significant. After transfection of 0C mimics, the expression of FUT4 was significantly downregulated. When transfected with FUT4 C DNA, the expression of FUT4 could be regulated by.Anti-miR-200c transfection, and the expression of FUT4 could be up to be up. The same result was obtained when the FUT4 Si RNA was co transfected. Inhibition of the activation of Wnt/ beta -catenin signaling pathway affects the function of endometrial cells. Transfection of miR-200c can inhibit the expression of p-GSK3 beta, beta -catenin, and the result is consistent with the effect of Wnt/ beta -catenin pathway inhibitor DKK. When the FUT4 C DNA co transfection is co transfected, it can reactivate the p-GSK3 beta, beta expression. The expression of 0C can activate the expression of p-GSK3 beta, beta -catenin and repress the expression of p-GSK3 beta and beta -catenin after CO transfection with FUT4 Si RNA. Conclusion: 1.miR-200c can be detected by serum, and its expression is negatively correlated with FUT4 and.2. inhibition miR-200c expression can be modified between human endometrium cells and embryonic cells. Targeting.4.miR-200c regulates the activation of Wnt/ beta -catenin signaling pathway by targeting FUT4, and inhibits the establishment of endometrial receptivity.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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