miR-182对奶山羊子宫内膜上皮细胞中HOXA10基因的靶向调控作用研究

发布时间：2018-01-19 21:18

本文关键词： miR-182 奶山羊子宫内膜上皮细胞 HOXA10　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：MiR-182对动物的生长和发育具有调控作用,而HOXA10在胚胎植入过程有着重要的调节功能,生物信息学分析表明,miR-182可能靶向调控HOXA10。因此研究miR-182对奶山羊子宫内膜上皮细胞(EEC)中HOXA10基因的调控作用,有助于进一步阐明胚胎着床的分子机理。本研究以关中奶山羊为研究对象,首先检测miR-182和HOXA10在容受期和非容受期子宫内膜的表达,然后分析miR-182对HOXA10的靶向调控作用,最后检测miR-182对EEC的调控作用,主要研究结果如下:1.利用RT-qPCR技术分别检测miR-182和HOXA10在容受期和非容受期子宫内膜的表达。结果表明,mi R-182和HOXA10在容受期子宫内膜的表达均极显著高于(P0.01)非容受期子宫内膜的表达。2.利用双荧光素酶报告载体、RT-qPCR以及Western blot等检测技术,检测mi R-182对HOXA10的调控作用。结果表明,将miRNA模拟物与HOXA10双荧光素酶报告载体共转后,发现miR-182 mimic组荧光素酶活性极显著低于NC对照组(P0.01);而突变型各组荧光素酶活性均差异不显著,表明miR-182可与HOXA10基因3′UTR区的靶位点结合,验证HOXA10为miR-182的靶基因。RT-qPCR以及Western blot结果显示,miR-182在mRNA水平和蛋白水平均抑制了HOXA10的表达。3.利用Western blot及细胞周期和凋亡检测技术检测miR-182对EEC的调控作用。将miR-182转入EEC中,发现miR-182并不调控奶山羊EEC的周期,但促进了奶山羊EEC的凋亡。进一步研究发现miR-182通过下调BCL-2蛋白的表达,上调Caspase-3和Fas蛋白的表达促进EEC的凋亡。mi R-182靶向调控HOXA10基因,在mRNA水平和蛋白水平下调了HOXA10的表达;miR-182促进奶山羊EEC的凋亡。
[Abstract]:MiR-182 regulates the growth and development of animals, while HOXA10 plays an important role in the process of embryo implantation, bioinformatics analysis shows that. MiR-182 may target HOXA10.Therefore, the regulation of miR-182 on HOXA10 gene in dairy goat endometrial epithelial cells was studied. In order to further clarify the molecular mechanism of embryo implantation, the expression of miR-182 and HOXA10 in the endometrium of Guanzhong dairy goat was detected at first. Then, the target regulation of HOXA10 by miR-182 was analyzed. Finally, the regulatory effect of miR-182 on EEC was detected. The main results are as follows: 1. The expression of miR-182 and HOXA10 in the endometrium of the receptive phase and the non-receptive phase were detected by RT-qPCR technique. The expression of miR-182 and HOXA10 in the endometrium of the receptive phase was significantly higher than that in the non-receptive endometrium. 2. Double luciferase was used to report the expression of miR-182 and HOXA10 in the endometrium. RT-qPCR and Western blot were used to detect the regulatory effect of miR-182 on HOXA10. The results showed that the luciferase activity of miR-182 mimic group was significantly lower than that of NC control group (P 0.01). However, the luciferase activity of mutant groups was not significantly different, indicating that miR-182 could bind to the target site of HOXA10 gene 3UTR region. The results of RT-PCR and Western blot showed that HOXA10 was the target gene of miR-182. MiR-182 inhibited the expression of HOXA10 at both mRNA and protein levels. Using Western. Blot, cell cycle and apoptosis detection techniques were used to detect the regulation of miR-182 on EEC. MiR-182 was transferred into EEC. It was found that miR-182 did not regulate the cycle of EEC in dairy goats, but promoted the apoptosis of EEC in dairy goats. Further studies showed that miR-182 down-regulated the expression of BCL-2 protein. Upregulating the expression of Caspase-3 and Fas protein promoted the apoptosis of EEC. Mi R-182 targeted HOXA10 gene. The expression of HOXA10 was down-regulated at the level of mRNA and protein. MiR-182 promotes the apoptosis of EEC in dairy goats.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

【相似文献】